Targeting Cytochrome P450 (CYP) 1B1 Enzyme with Four Series of A-Ring Substituted Estrane Derivatives: Design, Synthesis, Inhibitory Activity, and Selectivity

Article abstract of DOI:10.1021/acs.jmedchem.8b00907

Cytochrome P450 (CYP) 1B1 is involved in the bioactivation of procarcinogens and drug resistance. To obtain selective CYP1B1 inhibitors over CYP1A1, we synthesized four series of estrane derivatives: (1) 12 estrone (E1)- and 17β-estradiol (E2)-derivatives

Full text of DOI:10.1021/acs.jmedchem.8b00907

Subscriber access provided by Kaohsiung Medical University
Article
Targeting Cytochrome P450 (CYP) 1B1 Enzyme with
Four Series of A-ring Substituted Estrane Derivatives:
Design, Synthesis, Inhibitory Activity and Selectivity
Raphaël Dutour, Jenny Roy, Francisco Cortés-Benítez, Rene Maltais, and Donald Poirier
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Sep 2018
Downloaded from http://pubs.acs.org on September 14, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
J. Med. Chem. (revised)
Targeting Cytochrome P450 (CYP) 1B1 Enzyme with Four Series of Aꢀring
Substituted Estrane Derivatives: Design, Synthesis, Inhibitory Activity and
Selectivity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a,b
a
a,c
a
Raphaël Dutour , Jenny Roy , Francisco CortésꢀBenítez , René Maltais , and Donald
a,b,*
Poirier
a
Laboratory of Medicinal Chemistry, Endocrinology and Nephrology Unit, CHU de Québec –
Research Center, Québec, QC, G1V 4G2, Canada
b
Department of Molecular Medicine, Faculty of Medicine, Université Laval, Québec, QC, G1V 4G2,
Canada
c
Department of Pharmacy, Faculty of Chemistry, National Autonomous University of Mexico,
Mexico City, 04510, Mexico
(*) Corresponding Author:
Donald Poirier
Laboratory of Medicinal Chemistry
CHU de Québec – Research Center (CHUL, T4ꢀ42)
2
705 Laurier Boulevard
Québec, QC, G1V 4G2, Canada
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Tel.: 1ꢀ418ꢀ654ꢀ2296; Fax: 1ꢀ418ꢀ654ꢀ2298; Eꢀmail: donald.poirier@crchul.ulaval.ca
ABSTRACT: Cytochrome P450 (CYP) 1B1 is involved in the bioactivation of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
procarcinogens and drugꢀresistance. To obtain selective CYP1B1 inhibitors overs CYP1A1,
we synthesized four series of estrane derivatives: 1) twelve estrone (E1)ꢀ and 17βꢀestradiol
(E2)ꢀderivatives bearing a 3ꢀ or a 4ꢀpyridinyl core at C2, C3, or C4, 2) eight estrane
derivatives with different sulfur groups at C3, 3) nineteen E1ꢀ and E2ꢀderivatives bearing
distinct aryls at C2, and 4) five Dꢀring derivatives. E2ꢀderivatives were more active than
oxidized E1ꢀanalogs, thus highlighting the key role of 17βꢀOH for interaction with CYP1B1.
2ꢀ(4ꢀFluorophenyl)ꢀE2 was the best CYP1B1 inhibitor (IC50 = 0.24 µM) with a selectivity
index (SI) of 20 over CYP1A1. Furthermore, the addition of a C17αꢀethynyl group as Dꢀring
modification improved the SI to 25 with only a slight loss of activity (IC50 = 0.37 µM). Our
docking results showed that these compounds fit better into the CYP1B1 binding site than that
of CYP1A1.
KEYWORDS: Steroid, estrane derivatives, cytochrome P450, CYP1B1, enzyme inhibitor,
molecular docking
2
ACS Paragon Plus Environment

Page 3 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Cytochromes P450 (CYPs) constitute an essential family of hemoproteins involved in
1
ꢀ4
the metabolism of a wide variety of endogenic and xenobiotic compounds. The CYP1
family belongs to the eighteen CYP gene families known in humans, and includes three
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
,4
enzymes: CYP1A1, CYP1A2, and CYP1B1. CYP1B1 is an attractive therapeutic target for
different reasons: 1) it is involved in the bioactivation of several procarcinogens such as
benzo[α]pyrene, 2) it catalyzes the 4ꢀhydroxylation of 17βꢀestradiol (E2) subsequently
leading to the formation of E2ꢀ3,4ꢀquinone, a mutagenic compound able to bind DNA
covalently; and 3) it is associated with drugꢀresistance because it interacts with the
5ꢀ11
metabolism of some anticancer agents, such as docetaxel, paclitaxel, and cisplatin.
Furthermore, an overexpression of CYP1B1 has been observed in distinct types of human
cancers, such as breast, lung, esophagus, skin, testis, colon, lymph node, and brain. Therefore,
the use of a CYP1B1 inhibitor associated with an anticancer agent could be a promising
strategy to treat cancer cases in which CYP1B1 is overexpressed, and has developed drug
12,13
resistance.
14
In a recently published review, we reported the different CYP1B1 inhibitors
identified since 2003 among distinct families of chemicals: flavonoids, transꢀstilbenes,
coumarins, alkaloids, anthraquinones, and several other compounds. Flavonoid and stilbene
derivatives have been extensively studied for their ability to interact with CYP1 enzymes and
1
3ꢀ16
the best CYP1B1 inhibitors known to date belong to these two families of chemicals.
However, only two studies were conducted for CYP1B1 inhibition by steroid derivatives.
1
7,18
This is relatively surprising, given that some estraneꢀbased derivatives are substrates of this
19ꢀ24
enzyme. Having developed an expertise in the synthesis of steroid derivatives,
we
therefore decided to focus on this family of natural products, especially the C18ꢀ, C19ꢀ and
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C21ꢀsteroids, as potential CYP1B1 inhibitors. In an initial study, the inhibitory activity of a
collection of 90 steroid derivatives previously synthesized in our laboratory was evaluated on
CYP1B1 using the ethoxyresorufinꢀOꢀdeethylase (EROD) assay. This screening led to the
identification of 3ꢀthioestrone (IC50 = 3.4 ꢁM) as the most potent CYP1B1 inhibitor in this
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
25
series of steroid derivatives. In this respect, through molecular modeling studies, we
observed that the 3ꢀSH of 3ꢀthioestrone is closer (3.36 Å) to the iron atom of the CYP1B1
heme system than the 3ꢀOH of estrone (E1) and E2. These observations also suggest that
CYP1B1 inhibitory activity could be improved by introducing, a chemical group (pyridine,
26ꢀ30
triazole, etc.) on the Aꢀring of the C18ꢀsteroid core, known to interact with heme systems.
Otherwise, it should be emphasized that an estrane nucleus with a phenyl ring at C2
shares some structural similarity with that of αꢀnaphthoflavone (ANF) (Figures 1A and 1B), a
31
known potent CYP1B1 inhibitor which was coꢀcrystallized with CYP1B1. In this 3Dꢀ
structure, the phenyl moiety of ANF is oriented towards CYP1B1’s heme system iron atom.
By introducing different chemical groups on the phenyl moiety of ANF, Cui et al. identified
11
the most potent CYP1B1 inhibitor to date (3’ꢀFꢀ6,7,10ꢀtriꢀMeOꢀANF, Figure 1A).
Interestingly, the tricyclic core of this ANF derivative can be superimposed with the A/B/Cꢀ
rings of the steroidal scaffold (Figure 1C).
4
ACS Paragon Plus Environment

Page 5 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. (A) Chemical structures of αꢀnaphthoflavone (ANF), 3'ꢀFꢀ6,7,10ꢀtriꢀMeOꢀANF and 2ꢀPhꢀ
E1; (B) Molecular overlay of ANF (green) and 2ꢀPhꢀE1 (purple) cores; (C) Molecular overlay of 3'ꢀFꢀ
6,7,10ꢀtriꢀMeOꢀANF (yellow) and 2ꢀPhꢀE1 (purple) cores. The molecular overlays, as well as the
3
2
images, were produced using the BIOVIA Discovery Studio Visualizer. The molecular geometry of
compounds was previously optimized by the semiꢀempirical PM6 method using Gaussian 09
3
3
software.
Based on these observations and on docking studies, we synthesized three series of
estrane derivatives in order to identify potent CYP1B1 inhibitors (Figure 2). Series 1
regroups twelve E1 and E2 derivatives bearing a 3ꢀ or a 4ꢀpyridinyl moiety at C2, C3 or C4 of
the estrane core. Indeed, the nitrogen atom of pyridine is known to generate interactions with
26
the iron of heme systems. Some results obtained with this series of pyridinylꢀestrane
34
derivatives have been the subject of a preliminary report by our research group. It was
observed that estrane derivatives bearing the pyridinyl moiety at C2 were much more potent
CYP1B1 inhibitors than those with a pyridinyl at C3 or C4. Moreover, compounds bearing a
17βꢀOH exhibited better inhibitory effect than their oxidized form (17ꢀC=O). The best two
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CYP1B1 inhibitors of series 1 were the E2ꢀderivative with a 3ꢀpyridinyl at C2 (compound 1b)
and its counterpart with a 4ꢀpyridinyl at C2 (compound 2b).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Chemical structures of the three series of estrane derivatives designed and synthesized for
the inhibition of CYP1B1. Partial numbering of carbons (left structure) and steroid (AꢀD) ring
identification (right structure) are reported.
Herein, we report the chemical synthesis, characterization, and CYP1B1 inhibition
(EROD test) of additional estrane derivatives represented by series 2 and 3 (Figure 2). In
series 2, a sulfur group was introduced at C3 to promote an interaction with the heme group.
In series 3, the design of E1 and E2 derivatives was based on the similarity of estrane nuclei
with ANF and 3’ꢀFꢀ6,7,10ꢀtriꢀMeOꢀANF. Finally, we generated four Dꢀring derivatives of our
best CYP1B1 inhibitor, compound 20b, to reduce the metabolization of the 17βꢀOH group
and to see the impact on inhibitory activity. Selectivity of the inhibitors for CYP1B1 over
CYP1A1 was also assessed.
6
ACS Paragon Plus Environment

Page 7 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
RESULTS AND DISCUSSION
Synthesis of new estrane derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of estrane-sulfur derivatives (Series 2)
Based on the results of a first screening that identified 3ꢀthioꢀE1 as a weak CYP1B1
25
inhibitor, we synthesized eight new estrane derivatives bearing different sulfur groups at C3
(compounds 7aꢀb, 8aꢀb, 9aꢀb, and 10aꢀb, Scheme 1). Compound 7a, with a thiomethyl at C3,
was obtained in one step from 3ꢀthioꢀE1 by a methylation with methyl iodide in the presence
of cesium carbonate (Cs CO ) in refluxing acetonitrile (ACN). Thereafter, compound 7b was
2
3
obtained from 7a by a reduction of the C17 ketone with sodium borohydride (NaBH
4
) at 0 °C.
For this kind of unsubstituted Dꢀring steroid, the angular 18ꢀCH group provides an excellent
3
35ꢀ37
stereoselectivity and only the 17ꢀalcohol was observed.
a
Scheme 1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
3
Chemical synthesis of 3ꢀthioꢀestrane derivatives (Series 2). Reagents and conditions: (a) CH I,
Cs
2
CO
3
, ACN, reflux, 2 h; (b) NaBH
4
, MeOHꢀDCM (9:1), 0 °C, 2 h; (c) 3ꢀ(bromomethyl)pyridine
O (8:2), 0 °C, 2 h.
hydrobromide, Cs
2
CO , TEA, TBAI, ACN, reflux, overnight; (d) Oxone, MeOHꢀH
3
2
Compounds 8aꢀb are the E1 and E2 derivatives, respectively, with a pyridinꢀ3ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ylmethylthio group at C3 of the steroid core. Since pyridine is known to generate interactions
2
6
with heme sytems, we synthesized these compounds for a combined effect of the sulfur
atom and the pyridine moiety. Compound 8a was obtained from 3ꢀ(bromomethyl)pyridine
2 3
hydrobromide in refluxing ACN with Cs CO and triethylamine (TEA). A small quantity of
tetrabutylammonium iodide (TBAI) was also added to promote the reaction by replacing the
bromine atom of 3ꢀ(bromomethyl)ꢀpyridine by a more reactive iodine atom. Compound 8b
4
was next obtained by a reduction of 8a with NaBH .
Finally, we synthesized the sulfoxides 9aꢀb and the sulfones 10aꢀb because they are
highly probable metabolites of 7aꢀb due to the oxidizability of the sulfur atom. Compounds
9a and 10a were obtained in the same time from 7a using potassium peroxymonosulfate
2
(Oxone) as oxidizing agent. This reaction was performed in MeOHꢀH O at 0 °C and was
carefully monitored to avoid complete oxidation of 7a to 10a. Compounds 9b and 10b were
obtained in the same conditions used for the synthesis of 9a and 10a, but using 7b instead of
7
a as starting product.
Synthesis of 2-aryl-estrane derivatives (Series 3)
Based on the structural similarity between the steroid nucleus and ANF, as well as on
11
the good results obtained by Cui et al. when introducing small groups to the phenyl moiety
of ANF, we developed a third series of estrane derivatives with different aryl groups at C2 of
the steroid core (compounds 11aꢀb to 21a, Scheme 2). Phenyl groups bearing small chemical
functions were selected because a tooꢀlarge group could impair the insertion of the steroid
8
ACS Paragon Plus Environment

Page 9 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
derivative into the catalytic site of CYP1B1. Fluorine or chlorine atom was chosen in the
design of 12aꢀb, 13aꢀb, 14aꢀb, 15aꢀb, and 16aꢀb because ANF derivatives bearing halogens
11
on the phenyl core were found to be particulary potent CYP1B1 inhibitors. Phenol and
anisole derivatives 17aꢀb, 18a, 19a, and 20aꢀb were also prepared because it was observed
that the presence of hydroxyl or methoxy groups can increase the selectivity of inhibition
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13,16
towards CYP1B1.
The aniline derivative 21a was also prepared based on the potential
ability of the nitrogen atom to interact with the iron of heme groups. Aryl derivatives bearing
the same substituent, but at different positions (3ꢀ and 4ꢀ), were thus tested to complement our
structureꢀactivity relationship (SAR) study. Finally, 2ꢀphenylꢀestrane derivatives 11aꢀb were
added as references for this series of 2ꢀarylꢀestrane derivatives.
a
Scheme 2
a
Chemical synthesis of the 2ꢀarylꢀestrane derivatives (Series 3). Reagents and conditions: (a) Rꢀ
B(OH)
2
, Pd(dppf)Cl
2
3 4
, K PO , DMF, MW, 120 °C, 3ꢀ4 h; (b) 10 % HCl aq.ꢀMeOH (1:9), 50 °C,
overnight; (c) NaBH
4
, MeOHꢀDCM (9:1), 0 °C, 2 h.
The nineteen estrane derivatives of series 3 (compounds 11aꢀb to 21a) were
synthesized by reacting 2ꢀiodoꢀ3ꢀmethoxymethyl (MOM) etherꢀE1 with the corresponding
arylboronic acid in the same reaction conditions used for the synthesis of the series of
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pyridinylꢀestrane derivatives (Series 1). Reactions were performed at 120 °C in
dimethylformamide (DMF) under microwave irradiation with potassium phosphate tribasic
(
K
3
PO
4
)
as
base and
[1,1′ꢀbis(diphenylphosphino)ferrocene]dichloropalladium(II)
(Pd(dppf)Cl
2
) as catalyst. This Suzuki coupling reaction provided these 2ꢀarylꢀestrane
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
derivatives in good yields, but the presence of the MOM protecting group at C3 of the steroid
backbone was important. In fact, this ether group promotes the reaction by complexing the
palladium catalyst. After the Suzuki coupling, the MOM group was hydrolysed using
chlorhydric acid in methanol (MeOH) to obtain the eleven 2ꢀarylꢀE1 derivatives 11aꢀ21a.
Based on their CYP1B1 inhibitory activities, eight candidates were selected and the C17ꢀ
4
carbonyl was reduced with NaBH to obtain the 2ꢀarylꢀE2 derivatives 11bꢀ17b and 20b.
Synthesis of D-ring derivatives of 20b (compounds 23-26)
34
In our preliminary report on pyridinylꢀestrane derivatives (Series 1), we evaluated
the plasma concentration of 1b (Figure 2) in rats. We observed that the alcohol 1b was
oxidized at C17 to form the ketone 1a, and that both compounds 1aꢀb were eliminated after
6
h. To stabilize the 17βꢀOH functionality towards an oxidation by PhaseꢀI metabolism
enzymes as well as a glucuronidation or a sulfatation by PhaseꢀII metabolism enzymes, we
synthesized Dꢀring derivatives 23ꢀ26 (Scheme 3). The potent CYP1B1 inhibitor 20b (Series
3) was thus selected and modified by adding a small methyl, ethynyl or methoxy group
known to protect a 17βꢀOH group.
a
Scheme 3
10
ACS Paragon Plus Environment

Page 11 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Synthesis of Dꢀring derivatives of 20b. Reagents and conditions: (a) CH I, NaH, DMF, rt, overnight;
3
(
b) CH
3
I, Cs
2
CO
3
3
, ACN, reflux, 2ꢀ3 h; (c) CH MgI, toluene, 80 °C (4 h) to rt (overnight); (d) i. TMSꢀ
acetylene, MeLi, THF, rt, overnight; ii. K
2
CO , MeOH, rt, 5 h.
3
Compound 23 was synthesized from 20b by a diꢀOꢀmethylation of 3ꢀ and 17βꢀOH
groups, with methyl iodide in anhydrous DMF, and sodium hydride as base. The intermediate
compound 22 was synthesized from 20a by a methylation of the 3ꢀOH group with methyl
2 3
iodide and Cs CO in refluxing ACN, and was next used for the preparation of 24 and 25.
Compound 24 was obtained by adding a methyl group at position 17α of 22 using
methylmagnesium iodide in anhydrous toluene. Compound 25 was obtained from 22 through
an ethynylation at position 17α in two steps: 1) adding lithium trimethylsilylacetylide in
anhydrous tetrahydrofuran (THF) and 2) hydrolyzing the silylacetylenic intermediate. Finally,
compound 26 was obtained using the same reaction conditions reported for the synthesis of
25, but with 20a as starting product. This compound was prepared to evaluate the impact of
the methoxy group at C3 on the metabolic stability of these Dꢀring derivatives.
Assessment of estrane derivatives as CYP1B1 inhibitors
The inhibitory activities on CYP1B1 of newly synthesized estrane derivatives (Series
2 and 3; Figure 2) were evaluated using the standard EROD assay which is conventionally
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
used to assess CYP1 activity (Tables 1 and 2). These assays were performed with
recombinant human CYP1B1 enzyme and a NADPH regenerating system. The transformation
of resorufin ethyl ether by CYP1B1 into fluorescent resorufin was measured in this enzymatic
assay to evaluate CYP1B1 activity. ANF, a known potent CYP1B1 inhibitor, was used as
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
15,31
reference for these EROD assays.
CYP1B1 inhibitory activities of estrane-sulfur derivatives
Concerning the EROD assay results obtained with the eight thioꢀestrane derivatives as
CYP1B1 inhibitors (Table 1), only 8aꢀb have shown significant inhibitory activity against
CYP1B1. These two compounds bear the same pyridinꢀ3ꢀylmethanethio group at C3 and the
alcohol 8b (17βꢀOH) is more active than its oxidized homolog at C17, the ketone 8a. This
observation highlights the key role of 17βꢀOH for CYP1B1 inhibition and is in agreement
34
with the results obtained with the compounds of series 1. On the other hand, compounds 7aꢀ
b, with a thiomethyl group at C3 and their likely metabolites resulting from the oxidation of
the sulfur atom, compounds 9aꢀb and 10aꢀb, are very weak inhibitors. Considering these
results, the sulfur atom does not seem to play a major role in the inhibition of CYP1B1.
However, adding a pyridine moiety to the steroid core led to a significant gain in inhibitory
activity, as exemplified with compounds 8aꢀb. It should be noted that they are less potent
inhibitors than ANF (48 ± 3, 61 ± 3, and 94 ± 3% of inhibition at 0.3 ꢁM for 8a, 8b, and
ANF, respectively).
Table 1. Inhibition of CYP1B1 and CYP1A1 activities by a series of estraneꢀsulfur
derivatives (Series 2)
12
ACS Paragon Plus Environment

Page 13 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CYP1B1
CYP1B1
CYP1A1
Inhibition
(%) at 0.3
CYP1A1
Inhibition
(%) at 3
C3ꢀ
17ꢀO or 17βꢀ
OH
Inhibition
Inhibition
(%) at 3
a
Compd
substituent
(%) at 0.3
b
b
b
b
ꢁ
M
ꢁM
ꢁM
ꢁM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7a
7b
8a
8b
9a
9b
CH
3
3
S
S
O
17βꢀOH
O
14 ± 3
5 ± 1
34 ± 3
26 ± 5
87 ± 1
94 ± 1
57 ± 1
25 ± 2
7 ± 1
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
CH
3ꢀPyrCH
2
2
S
S
48 ± 3
61 ± 3
16 ± 3
10 ± 2
7 ± 4
9 ± 9
11 ± 2
ꢀꢀ
36 ± 5
40 ± 8
ꢀꢀ
3ꢀPyrCH
17βꢀOH
O
CH
3
3
SO
SO
CH
17βꢀOH
O
ꢀꢀ
ꢀꢀ
1
0a
0b
CH
3
SO
2
2
ꢀꢀ
ꢀꢀ
1
CH
3
SO
17βꢀOH
ꢀ
0 ± 3
5 ± 3
ꢀꢀ
ꢀꢀ
ANF
ꢀ
94 ± 3
101 ± 1
30 ± 3
75 ± 6
a
b
See Scheme 1 for the structure of these compounds.
Inhibition of the transformation of resorufin ethyl ether into resorufin by human CYP1B1 or
CYP1A1 in the presence of NADPH. Two experiments performed in triplicate (±SD).
CYP1B1 inhibitory activities of 2-aryl-estrane derivatives
The results obtained with compounds from series 1 and 2, and the superimposition of
the estrane nucleus with the best CYP1B1 inhibitor of the ANF family (Figure 1C), oriented
our work toward the synthesis of a third series of estrane derivatives bearing different phenyl
moieties at C2 (compounds 11aꢀb to 21a, Scheme 2).
As a first important observation, we can see that almost all compounds of this third
series of estrane derivatives are potent CYP1B1 inhibitors with inhibition percentages that are
higher than 68 % at 0.3 ꢁM and a full inhibition of CYP1B1 activity at 3 ꢁM (Table 2).
Indeed, only compounds 18a, 19a, and 21a, with a 4ꢀhydroxyphenyl, a 3ꢀmethoxyphenyl, and
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a 3ꢀaminophenyl moiety, respectively, at C2 of the E1 core, have shown weak inhibitory
activities towards CYP1B1 (EROD assay). Another important point is that the ketone 11a,
with a phenyl group at C2 of the steroid core, showed a better CYP1B1 inhibition than those
observed with E1ꢀderivatives 1a and 2a, bearing respectively a 3ꢀ and a 4ꢀpyridinyl moiety at
position 2. Thus, the introduction of a C2ꢀphenyl ring bearing small chemical groups is a good
strategy to optimize the inhibition of estrane derivatives towards CYP1B1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Inhibition of CYP1B1 activity by a series of 2ꢀarylꢀestrane derivatives (Series 3)
CYP1B1
Inhibition
(%) at 0.3
CYP1B1
Inhibition
(%) at 3
CYP1A1
Inhibition
(%) at 0.3
CYP1A1
Inhibition
(%) at 3
a
Compd
R
X
b
b
b
b
ꢁM
ꢁM
ꢁM
ꢁM
c
1a
1b
3ꢀPy
3ꢀPy
O
17βꢀOH
O
38 ± 2
90 ± 2
100 ± 1
55 ± 11
102 ± 1
101 ± 2
101 ± 1
101 ± 1
101 ± 1
101 ± 1
103 ± 1
ꢀꢀ
ꢀꢀ
c
85 ± 1
53 ± 4
ꢀꢀ
77 ± 2
ꢀꢀ
c
2a
4ꢀPy
21 ± 3
c
2b
4ꢀPy
17βꢀOH
O
87 ± 1
5 ± 4
5 ± 5
9 ± 10
3 ± 2
12 ± 5
2 ± 1
3 ± 1
14 ± 11
16 ± 2
45 ± 1
23 ± 3
48 ± 7
8 ± 1
0 ± 4
11a
11b
12a
12b
Ph
69 ± 3
82 ± 2
67 ± 7
88 ± 1
71 ± 2
91 ± 2
Ph
17βꢀOH
O
3ꢀFꢀPh
3ꢀFꢀPh
4ꢀFꢀPh
4ꢀFꢀPh
17βꢀOH
O
13a
13b
17βꢀOH
14
ACS Paragon Plus Environment

Page 15 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
4a
4b
5a
5b
6a
6b
7a
7b
3,4ꢀdiꢀFꢀPh
O
77 ± 2
92 ± 2
74 ± 1
91 ± 1
79 ± 2
96 ± 2
92 ± 2
96 ± 1
19 ± 1
48 ± 4.5
95 ± 2
97 ± 1
36 ± 4
91 ± 2
100 ± 1
100 ± 1
100 ± 2
100 ± 1
101 ± 2
99 ± 3
1 ± 1
2 ± 1
5 ± 4
16 ± 8
9 ± 10
12 ± 10
10 ± 1
10 ± 3
ꢀꢀ
15 ± 20
47 ± 23
11 ± 4
51 ± 13
5 ± 5
1
3,4ꢀdiꢀFꢀPh 17βꢀOH
1
3ꢀClꢀPh
3ꢀClꢀPh
O
1
17βꢀOH
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4ꢀClꢀPh
O
1
4ꢀClꢀPh
17βꢀOH
63 ± 6
39 ± 13
57 ± 3
ꢀꢀ
1
3ꢀHOꢀPh
3ꢀHOꢀPh
4ꢀHOꢀPh
3ꢀMeOꢀPh
4ꢀMeOꢀPh
O
103 ± 1
100 ± 1
30 ± 2
1
17βꢀOH
18a
19a
20a
O
O
O
81 ± 1
ꢀꢀ
ꢀꢀ
103 ± 1
100 ± 2
55 ± 3
8 ± 10
14 ± 7
ꢀꢀ
18 ± 9
51 ± 12
ꢀꢀ
2
0b
1a
ANF
4ꢀMeOꢀPh 17βꢀOH
2
3ꢀH
2
NꢀPh
O
ꢀ
ꢀ
104 ± 1
30 ± 3
75 ± 6
a
b
See Scheme 2 and Figure 2 for the structures of these compounds.
Inhibition of the transformation of resorufin ethyl ether into resorufin by human CYP1B1 or
CYP1A1 in the presence of NADPH. Two or three experiments performed in triplicate (±SD).
c
Data from reference 34.
The second major observation is a very interesting gain in CYP1B1 inhibitory activity
observed at 0.3 µM for all E2 derivatives (17βꢀOH), in comparison with their oxidized
homologs (E1 derivatives) with a ketone at C17. These results correlate with those previously
obtained with series 1 and 2, thus confirming the key role that this hydroxy function at C17
plays, by promoting the interaction of estrane derivatives with the catalytic site of CYP1B1.
From the results reported in Table 2, the best candidates are compounds 13b, 14b,
1
5b, 16b, 17aꢀb, and 20aꢀb with CYP1B1 inhibitory activities equal or superior to that of
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ANF (91 ± 2% of inhibition at 0.3 ꢁM). Compound 13b, with a 4ꢀfluorophenyl core at C2,
had the same CYP1B1 inhibitory profile as its analog, compound 14b, with a 3,4ꢀ
difluorophenyl moiety (91 ± 2 and 92 ± 2% of inhibition at 0.3 ꢁM, respectively). Compound
12b, bearing a 3ꢀfluorophenyl group, was slightly less active than 13b and 14b, suggesting
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
that the 4ꢀposition on the phenyl group at C2 appears to be the best for the interaction of the
fluorine atom with the heme of CYP1B1. Compounds 15b and 16b, with a 3ꢀ and a 4ꢀ
chlorophenyl moiety at C2 of the steroid core, respectively, showed close CYP1B1 inhibition
potentials (91 ± 1 and 96 ± 2% of inhibition at 0.3 ꢁM, respectively), but the 4ꢀposition seems
to be slightly advantageous for the chlorine atom. Moreover, compound 16b, with a 4ꢀ
chlorophenyl group at C2, was more active than its 4ꢀfluoro homolog, 13b. This result
suggests that the chlorine atom is a better substituent than fluorine for CYP1B1 inhibition.
Compounds 17a and 17b are the E1ꢀ and E2ꢀderivatives with a 3ꢀhydroxyphenyl core
at C2 (92 ± 2 and 96 ± 1% of CYP1B1 inhibition at 0.3 ꢁM, respectively). Similarly, 20a and
20b are 4ꢀmethoxyphenyl derivatives (95 ± 2 and 97 ± 1% of inhibition at 0.3 ꢁM,
respectively). If we compare the results obtained for the compounds from series 3, these four
chemicals show higher inhibition percentages than those of ANF and close to 100% when
tested at 0.3 µM. Compounds 17b and 20b are also the most active CYP1B1 inhibitors of
series 3 but also of the three series of estrane derivatives tested (96 ± 1 and 97 ± 1% of
inhibition at 0.3 ꢁM, respectively).
Furthermore, we can see a significant difference in CYP1B1 inhibitory activity for 17a
(3ꢀhydroxyphenyl) and 20a (4ꢀmethoxyphenyl at C2) in comparison with their respective
homologs 18a (4ꢀhydroxyphenyl) and 19a (3ꢀmethoxyphenyl), bearing the same substituent
on the phenyl group at C2, but at a distinct position. We observed that the 3ꢀposition is more
advantageous for the hydroxy function (17a vs 18a) while the 4ꢀposition is preferable for the
methoxy substituent (19a vs 20a). Thus, the position of the hydroxy and methoxy functions
16
ACS Paragon Plus Environment

Page 17 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
on the phenyl moiety at C2 appears to have a stronger impact on their activity than the
position of halogenated substituents.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CYP1B1 inhibitory activities of D-ring derivatives (Series 4)
The formation of a 17βꢀmethoxy derivative (compound 23) or a tertiary alcohol
(compounds 24ꢀ26) is a known strategy to stabilize the steroidal C17ꢀsecondary alcohol or
C17ꢀketone towards Phase I and Phase II metabolism. The impact of these small
modifications on CYP1B1 inhibition was consequently assessed by testing their inhibitory
activities on CYP1B1 (Table 3). The results showed that the 17βꢀOMe derivative 23 did not
inhibit CYP1B1 (20% at 3 µM), whereas the 17αꢀmethylation and 17αꢀethynylation
(compounds 24 and 25, respectively) were less detrimental for the inhibition of CYP1B1 (46
and 55% at 3 µM, respectively). However, the presence of a MeO group at C3 instead of the
OH group, a change that would have been helpful to stabilize the phenolic Aꢀring, also
reduced the inhibitory potency at 0.3 µM from 77 to 38% for 20a and 22, respectively. A dual
modification at C3 (Oꢀmethylation) and C17α (ethynylation) was detrimental to CYP1B1
inhibition, but the 17αꢀethynylation alone of compound 20b, producing the tertiary alcohol
26, allowed to keep very good levels of enzyme inhibition (58% and 94% at 0.3 and 3 µM,
respectively).
Table 3. Inhibition of CYP1B1 activity by Dꢀring derivatives (Series 4)
a
Compd
C2ꢀ
C3ꢀ
C17β/17αꢀ
CYP1B1
CYP1B1
CYP1A1
CYP1A1
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Substituent Substituent Substituent
Inhibition
Inhibition
(%) at 3
Inhibition
(%) at 0.3
Inhibition
(%) at 3
(%) at 0.3
b
b
b
b
µM
µM
µM
µM
2
2
2
2
2
3
4
5
6
2
4ꢀCH
4ꢀCH
4ꢀCH
4ꢀCH
4ꢀCH
4ꢀCH
4ꢀCH
3
3
3
3
3
3
3
OꢀPh
OꢀPh
OꢀPh
OꢀPh
OꢀPh
OꢀPh
OꢀPh
CH
CH
CH
3
3
3
O
O
O
OCH
3
/ H
11 ± 4
8 ± 8
20 ± 1
46 ± 7
55 ± 2
94 ± 1
79 ± 1
85 ± 2
107 ± 1
104 ± 1
1 ± 1
2 ± 1
4 ± 3
7 ± 5
5 ± 6
6 ± 4
9 ± 1
23 ± 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH / CH
3
OH / C≡CH
OH / C≡CH
= O
6 ± 3
HO
58 ± 1
38 ± 1
77 ± 1
97 ± 1
93 ± 1
c
c
CH
3
O
1.2
4.7
20a
HO
= O
8 ± 10
14 ± 7.2
30 ± 3
18 ± 9
51 ± 12
75 ± 6
20b
HO
ꢀꢀ
OH / H
ꢀꢀ
ANF
ꢀꢀ
a
b
See Schemes 1 and 3 for the structures of these compounds.
Inhibition of the transformation of resorufin ethyl ether into resorufin by human CYP1B1 or
CYP1A1 in the presence of NADPH. Two experiments performed in triplicate (±SD).
c
One experiment performed in triplicate.
Assessment of estrane derivatives as CYP1A1 inhibitors
CYP1A1 is mainly associated with the formation of 2ꢀOHꢀE2 contrary to CYP1B1,
which is mainly involved in the formation of mutagenic 4ꢀOHꢀE2. Therefore, to reduce the
ratio 4ꢀOHꢀE2 / 2ꢀOHꢀE2, a CYP1B1 inhibitor should ideally not inhibit CYP1A1. The most
potent CYP1B1 inhibitors from series 1 (compounds 1b and 2b), series 2 (compounds 8a and
8b), series 3 (compounds 11aꢀb to 17aꢀb and 20aꢀb) and Dꢀring derivatives (compounds 23ꢀ
2
6) were tested as CYP1A1 inhibitors using the EROD assay (Tables 1ꢀ3). The only
differences with the CYP1B1 assay were the use of less CYP1A1 (0.13 pmol instead of 0.7
pmol) and a shorter reaction time (25 min instead of 45 min). As reported in Table 1, the best
18
ACS Paragon Plus Environment

Page 19 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CYP1B1 inhibitor of series 2, the estraneꢀsulfur derivative 8b, showed a moderate selectivity
of inhibition for CYP1B1 over CYP1A1, as similarly observed for ANF. The keto derivative
8a provided better selectivity, but this compound only weakly inhibited CYP1B1. In the
compounds of series 1, the 4ꢀpyridinyl derivative 2b is more selective for CYP1B1 than the 3ꢀ
pyridinyl derivative 1b (Table 2). For series 3, sixteen of the nineteen arylꢀestrane derivatives
were tested and found to be selective for CYP1B1 over CYP1A1. At the higher
concentrations tested (3 µM), their inhibition levels ranged from 1 to 78% for CYP1A1,
whereas they fully inhibited CYP1B1, thus suggesting a very good selectivity for most of
these 2ꢀarylꢀestrane derivatives. The most selective compound from series 3 is clearly the 4ꢀ
fluoroꢀphenyl derivative 13b, which did not significantly inhibit CYP1A1 at 0.3 and 3 µM.
As reported in Table 3, compounds with a Dꢀring modification inhibited weakly CYP1A1,
but they are also weak CYP1B1 inhibitors. The ethynylated compound 26 was however
selected because it provided the best compromise between inhibition of CYP1B1 and nonꢀ
inhibition of CYP1A1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Selectivity of CYP1B1 inhibition over CYP1A1
The results obtained from the screening of estrane derivatives on both CYP1B1 and
CYP1A1 allowed us to select the best representative compounds of series 1ꢀ4. Compounds
2
b, 8b, 13b, and 26 were thus tested at several concentrations, and the IC50 values were
determined from the curve reported in Figures 3 and 4. The IC50 values ranged from 0.24 to
.54 µM for CYP1B1 and from 3.4 to 9.2 µM for CYP1A1, which values provided a good to
very good selectivity index (SI) ranging from 6 to 25 (Table 4). The estraneꢀsulfur derivative
b is the less potent and selective CYP1B1 inhibitor, as suggested by the screening results.
0
8
The 4ꢀpyridinylꢀ and 4ꢀfluoroꢀphenylꢀestrane derivatives 2b and 13b produced the same
inhibition of CYP1B1, but 13b is more selective (SI = 16 and 20, respectively). Interestingly,
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound 26, with a Dꢀring modification (17αꢀethynyl), provided the best selectivity (SI =
25) for CYP1B1 among these four compounds, and it is also more selective than the reference
ANF (SI = 11).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Curves of CYP1B1 (A) and CYP1A1 (B) inhibition obtained with E2 derivatives 2b, 8b,
13b, and 25. Each curve is representative of two experiments performed in triplicate.
Table 4. Selectivity of the inhibition for CYP1B1 over CYP1A1
b
Series
Cpd
Name
CYP1B1
CYP1A1
IC50 (µM)
3.9 ± 1.9
3.4 ± 2.6
SI
a
a
IC50 (µM)
1
2
2b
8b
2ꢀ(4ꢀpyridinyl)ꢀE2
3ꢀ(pyridineꢀ3ꢀ
0.24 ± 0.05
0.54 ± 0.08
16
6
ylmethanethio)ꢀE2
2ꢀ(4ꢀfluoroꢀphenyl)ꢀE2
2ꢀ(4ꢀmethoxyꢀphenyl)ꢀ
3
4
13b
26
0.24 ± 0.04
0.37 ± 0.09
4.9 ± 0.6
9.2 ± 3.5
20
25
17αꢀethynylꢀE2
ꢀꢀ
ANF nonsteroidal inhibitor
0.24 ± 0.08
2.7 ± 2.3
11
20
ACS Paragon Plus Environment

Page 21 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
b
Selectivity index (SI) = IC50 (CYP1A1) / IC50 (CYP1B1).
Two experiments performed in triplicate (±SEM)
Docking results
To investigate the potential binding modes of 2b, 8b, 13b, and 26, as well as their
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
selectivity, we carried out docking simulations using GOLD 5.4 software and the Xꢀray
structure of CYP1B1 (PDB ID: 3PM0) and CYP1A1 (PDB ID: 4I8V). For comparison
purposes, the structure of ANF was also docked. The first interesting observation for the
molecules screened in this study (Table 5) is that they displayed better GoldScore (GS) and
ChemPLP fitness score (CFS) for CYP1B1 than CYP1A1 (except for 8b). For instance, the
best docked conformation of ANF fit better into the binding site of CYP1B1 (GS = 69.8 and
CFS = 95.5) than CYP1A1 (GS = 69.6 and CFS = 87.1). These results showed the same
tendency as our experimental data against both isoenzymes.
Table 5. Docking scores for 2b, 8b, 13b, and 26 at the binding site of CYP1B1 and
a
CYP1A1 enzymes
Compound
CYP1B1
GS
CYP1A1
GS
CFS
89.4
102.2
91.7
96.0
95.5
CFS
90.7
108.2
75.3
65.7
87.1
2b
54.5
46.2
8b
64.2
63.7
13b
56.7
40.1
2
6
22.6
16.3
ANF
69.8
69.6
a
For GoldScore (GS) and ChemPLP fitness score (CFS) definitions, see Supporting
Information.
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The binding mode of 2b into CYP1B1 (GS = 54.5 and CFS = 89.4) and CYP1A1 (GS
= 46.2 and CFS = 90.7) differs considerably, since the steroidal scaffold is inversely oriented
for each enzyme (Figure S1, Supporting Information). However, it is important to note that
the pyridine ring at C2 is oriented towards the heme group without forming nitrogenꢀiron
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
34
bonds as performed by 1b. The most important interactions of 2b with the binding site of
CYP1B1 include: an Hꢀbond of 17βꢀOH with Asnꢀ228 (distance = 1.7 Å); piꢀstacking
interactions with residues Pheꢀ134, Pheꢀ231 and Glyꢀ329; a piꢀcation interaction between the
pyridine ring and the iron atom of the heme group; as well as weak hydrophobic bonds with
Leuꢀ509, Pheꢀ268, Alaꢀ133 and Alaꢀ330. On the contrary, the docked structure of 2b in
CYP1A1 interacts by means of: two Hꢀbonds between 17βꢀOH and 3ꢀOH with Serꢀ116
(distance = 1.7 Å) and Aspꢀ320 (distance = 2.0 Å), respectively; piꢀinteractions with Pheꢀ224,
Glyꢀ316 and the heme group as well. Nevertheless, we found that the orientation of 18ꢀCH of
3
2b promotes clashing into both enzymes, suggesting that this group may disrupt the correct
binding of this derivative. However, we assume that 2b fit better into the binding site of
CYP1B1 because there is just one bump with Glnꢀ332, instead of two formed with residues of
Ileꢀ115 and Pheꢀ224 into the cavity of CYP1A1.
Unlike 2b, the binding mode of 8b showed the same orientation into the active sites of
CYP1B1 (GS = 64.2 and GFS = 102.2) and CYP1A1 (GS = 63.7 and 108.2) (Figure S2,
Supporting Information). The 17βꢀOH group for both 8b structures is positioned to perform
Hꢀbonds with Asnꢀ265 (distance = 2.6 Å) and Asnꢀ255 (distance = 2.0 Å) for CYP1B1 and
CYP1A1, respectively. Moreover, these structures form piꢀinteractions with the side chain of
residues Phe and Gly as well as hydrophobic contacts with residues Ala, Leu and Val.
Surprisingly, 8b produced a piꢀinteraction between its Aꢀring and the aromatic ring of Pheꢀ
231 into the cavity of CYP1B1, whereas this interaction is absent with CYP1A1 because Pheꢀ
22
ACS Paragon Plus Environment

Page 23 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
24 residue is more distanced from the Aꢀring of 8b. In addition, as observed for 2b, the 18ꢀ
CH group of 8b exhibited steric hindrance with the aliphatic side chain of Ileꢀ115 into
3
CYP1A1, but this was not the case with CYP1B1. Thus, 8b fits better into CYP1B1,
providing slighty high GS than that obtained for CYP1A1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As observed for 8b, the steroidal core of 13b is positioned in the same way at the
binding site of both enzymes (Figure 4). Interestingly, we found improved GS, as well as
CFS scores for CYP1B1 (GS = 56.7 and CFS = 91.7) than CYP1A1 (GS = 40.1 and CFS =
7
5.3), suggesting better selectivity. The 4ꢀfluorophenyl ring attached to C2 of 13b is oriented
towards the heme group into the cavity of both isoenzymes. Curiously, 13b possesses the
34
same binding mode than that found with pyridinylꢀestrane CYP1B1 inhibitors 2b and 1b.
According to our docking results, 13b produces Hꢀbonds between 17βꢀOH and Asn residues
such as Asnꢀ228 (distance = 1.8 Å) and Asnꢀ222 (distance = 1.9 Å) into the binding site of
CYP1B1 and CYP1A1, respectively. Furthermore, the steroidal core interacts with
hydrophobic moieties of Val, Ala and Phe. Conversely, since the aromatic ring at C2 of 13b is
closer to the iron atom of CYP1A1 than CYP1B1, we found that this group promotes piꢀ
cation interactions with the heme group of CYP1A1 instead of piꢀpi Tꢀshaped interactions
with the pyrrole subunits of porphyrin as performed into CYP1B1. Nevertheless, we believe
that 13b exibited better GS and CFS for CYP1B1 because its Aꢀring produces more piꢀ
interactions in comparison with CYP1A1. These include piꢀpi Tꢀshaped with Pheꢀ231 and Piꢀ
sigma with Alaꢀ330.
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. (A) Binding mode of 13b docked at the binding site of CYP1B1 (light blue) (PDB 1D:
3PMO) and CYP1A1 (khaki) (PDB 1D: 418V). Yellow sticks represent 13b docked into the active site
cavity of CYP1B1, while purple sticks represent 13b docked into the active site cavity of CYP1A1.
Black sticks represent the heme group for both enzymes, whereas the iron atom is highlighted as a red
sphere. (B) 2D diagram of docked 13b in CYP1B1 and CYP1A1 showing the interactions with the
residues at the binding site.
The binding mode of 26 is oriented oppositely to its counterparts 2b, 8b, and 13b
(Figure 5). For instance, the 17βꢀOH group is oriented towards the heme group instead of
forming Hꢀbonds with Asn residues. On the other hand, the steroid core of 26 displays
hydrophobic contacts with Phe, Ile and Ala residues, as well as the heme group. In addition,
the Aꢀring performs piꢀinteractions with Phe and Gly residues. Moreover, the aromatic ring at
C2 is oriented towards the side chain of Pheꢀ268 and Pheꢀ258 residues of CYP1B1 and
3
CYP1A1, respectively, forming piꢀpi Tꢀshaped interactions. Interestingly, the 18ꢀCH of 26
has a negative interaction with Leuꢀ496 into CYP1A1, whereas a CH of the methoxyphenyl
moiety at C2 bumps with Leuꢀ264 into CYP1B1. Unexpectedly, the 3ꢀOH group of 26 forms
a Hꢀbond (distance = 2.4 Å) with the amide residu of Asnꢀ265 into the binding site of
24
ACS Paragon Plus Environment

Page 25 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CYP1B1 but not with Asnꢀ255 of CYP1A1. Therefore, this key interaction promotes a better
fit into the active site cavity of CYP1B1, and is the main reason why we found much better
GS (22.6) and CFS (96.0) than those of CYP1A1 (GS = 16.3 and CFS = 65.7). In fact, the
experimental data showed that CYP1B1 inhibition was notably improved by replacing the 3ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
CH O group of 25 by the 3ꢀOH of 26, thus supporting that the Hꢀbond donor group of 26 may
interact with Asnꢀ265. It is worth mentioning that, since the C17αꢀethynyl moiety is a rigid
group, it did not allow the correct interaction of the 17βꢀOH group with Asnꢀ228 residue as
performed by 2b and 13b. Thus, we assume that this group promotes the inversed orientation
of 26, also giving a slightly negative impact in its inhibitory effect against CYP1B1.
Figure 5. (A) Binding mode of 26 docked at the binding site of CYP1B1 (light blue) (PDB 1D:
3PMO) and CYP1A1 (khaki) (PDB 1D: 418V). Magenta sticks represent 26 docked into the active site
cavity of CYP1B1 while cyan sticks represent 26 docked into the active site cavity of CYP1A1. Black
sticks represent the heme group for both enzymes, whereas the iron atom is highlighted as a red
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sphere. (B) 2D diagram of docked 26 in CYP1B1 and CYP1A1, showing the interactions with the
residues at the binding site.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
We synthesized four series of new estrane derivatives in order to assess their
inhibitory activity towards CYP1B1 and CYP1A1: 1) twelve E1 and E2 derivatives bearing a
3
ꢀ or 4ꢀpyridinyl core at positions 2, 3 or 4 of the steroid nucleus (Series 1), eight estrane
derivatives with different sulfur groups at C3 (Series 2), nineteen 2ꢀarylꢀestrane derivatives
Series 3), and four Dꢀring derivatives (Series 4). These steroid derivatives were obtained in
(
1
13
good to very good yields, and were fully characterized by IR, H NMR, C NMR, and HRMS
analyses.
The results obtained with series 1 highlighted a very interesting correlation between
docking studies and the inhibitory activities measured for this series of pyridinylꢀestrane
derivatives, showing that position 2 is the best for CYP1B1 inhibition. With its 4ꢀpyridinyl
group at C2, compound 2b is a potent and selective CYP1B1 inhibitor. Compounds of series
2
were found to be less potent CYP1B1 inhibitors among the estrane derivatives tested in this
study. Only compounds 8aꢀb, with a pyridinꢀ3ꢀylmethanethio group, showed significant
inhibitory activities in this series of 3ꢀthioꢀestrane derivatives.
The design of the compounds in series 3 was based on the superimposition of ANF
and the steroid core of E1 and E2. The results were very promising, because almost all the 2ꢀ
arylꢀestrane derivatives were potent CYP1B1 inhibitors. Thus, position 2 of the estrane
nucleus appears to be the best one to introduce chemical groups such as aryl and pyridinyl
moieties. The most potent CYP1B1 inhibitors of this series are compounds 13b and 20b,
bearing a 4ꢀfluorophenyl and a 4ꢀmethoxyphenyl group at C2, respectively. As observed for
26
ACS Paragon Plus Environment

Page 27 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
series 1 and 2, compounds of series 3 with a hydroxy function at C17 (17βꢀOH) were more
potent CYP1B1 inhibitors than their oxidized homologs (ketone at C17). This conclusion
highlights the key role of 17βꢀOH for CYP1B1 inhibition by the formation of an Hꢀbond
between 17βꢀOH and the Asnꢀ228 residue. This secondary alcohol (17βꢀOH) was also
stabilized against the metabolism by adding a 17αꢀethynyl group, a Dꢀring modification that
increased the selective inhibition of CYP1B1 over CYP1A1, as exemplified by compound 26,
but induced an inversion of its binding mode, contrary to other estrane derivatives 2b, 8b, and
13b.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Our docking results showed that these new estrane derivatives fit better into the
CYP1B1 binding site than that of CYP1A1; correlating with our experimental data.
Moreover, we found that Asn residues play a critical role by forming Hꢀbonds with the 17βꢀ
OH and 3ꢀOH of the estrane derivatives into the active site cavity of both isoenzymes.
However, piꢀinteractions and unfavorable bumps with the steroid core are also crucial to the
inhibitory activity and selectivity of these novel steroid derivatives. Finally, it should be noted
that a small chemical modification in the structure of these estrane derivatives is enough to
induce a different orientation of the compound into the catalytic site of CYP1B1 or CYP1A1.
EXPERIMENTAL SECTION
General
Chemical reagents were purchased from SigmaꢀAldrich Canada Ltd. (Oakville, ON,
Canada). Dry dichloromethane (DCM) and dimethylformamide (DMF) were obtained from
SigmaꢀAldrich. Acetonitrile (ACN), ethyl acetate (EtOAc), hexanes and methanol (MeOH)
were obtained from Fisher Scientific (Montreal, QC, Canada) and were used as received.
Reactions using microwave irradiations were performed with a Biotage Initiator (Charlotte,
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 59
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NC, USA). Thinꢀlayer chromatography (TLC) and flashꢀcolumn chromatography were
performed on 0.20 mm silica gel 60 F254 plates (E. Merck; Darmstadt, Germany) and with
230–400 mesh ASTM silica gel 60 (Silicycle, Québec, QC, Canada), respectively. The purity
of final compounds to be tested was determined with a Shimadzu HPLC apparatus (Kyoto,
Japan) using a Shimadzu SPDꢀM20 photodiode array detector, an Alltima HP C18 column
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(250 mm x 4.6 mm, 5 ꢁm), and a solvent gradient of MeOH:water to MeOH (100%). The
wavelength of the UV detector was selected between 190 and 220 nm. All final compounds
showed a purity ≥ 95% (95.2 – 99.9%; 23 compounds) except for compounds 7a, 8a, 9b, 12aꢀ
b, 15aꢀb, 16b, and 21a (89.5 – 94.6%, 9 compounds). Infrared (IR) spectra were recorded on
a MB 3000 ABB FTIR spectrometer (Quebec, QC, Canada), and only the significant bands
ꢀ1
are reported in cm . Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz
1
13
for H and 100.6 MHz for C on a Bruker Avance 400 digital spectrometer (Billerica, MA,
USA). The chemical shifts (δ) were expressed in ppm and referenced to chloroform (7.26 and
77.0 ppm), dimethylsulfoxide (2.49 and 39.5 ppm) or methanol (3.31 ppm and 49.0 ppm) for
1
13
H and C NMR, respectively. Lowꢀresolution mass spectra (LRMS) were recorded on a
Shimadzu apparatus (Kyoto, Japan) equipped with an atmospheric pressure chemical
ionization source. Highꢀresolution mass spectra (HRMS) were provided by Pierre Audet at the
Chemistry Department of Université Laval (Quebec, QC, Canada).
Synthesis of estraneꢀsulfur derivatives (Series 2: compounds 7a,b to 10a,b)
3ꢀThiomethylꢀestraꢀ1,3,5(10)ꢀtrieneꢀ17ꢀone (7a)
To a solution of 3ꢀthioꢀestrone (100 mg, 0.35 mmol) and cesium carbonate (Cs CO )
2
3
(228 mg, 0.7 mmol) in ACN (15 mL) was added methyl iodide (MeI) (144 ꢁL, 2.8 mmol).
The resulting mixture was stirred and heated under reflux for 2 h. After cooling, the reaction
mixture was poured into water and extracted with EtOAc. The organic phase was washed with
28
ACS Paragon Plus Environment

Page 29 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
water, dried over magnesium sulfate (MgSO ), filtered, and evaporated under reduced
4
pressure. The crude compound was purified by flash chromatography with hexanes/EtOAc
(8:2) as eluent to give compound 1a as a white amorphous solid (70 mg, 67%). IR (KBr) υ:
1
1
736 (C=O); H NMR (CDCl
3
) δ: 0.91 (s, CH
3
ꢀ18), 1.40ꢀ2.45 (m, residual CH and CH
2
), 2.47
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
s, CH
3
S), 2.51 (dd, J
1
= 8.8 Hz, J
2
= 19.0 Hz, 16βꢀCH), 2.89 (m, CH
2
3
ꢀ6), 7.02 (s, CHꢀ4),
1
7
1
.07 (dd, J = 8.2 Hz, J = 1.9 Hz, CHꢀ2), 7.22 (d, J = 8.2 Hz, CHꢀ1); C NMR (CDCl ) δ:
1
2
3
3.8, 16.1, 21.5, 25.7, 26.4, 29.3, 31.5, 35.8, 38.1, 44.1, 47.9, 50.4, 124.5, 125.9, 127.4, 135.3,
+
136.9, 137.2, 220.8; HRMS for C19
purity: 92.4%.
H
25OS [M + H] : calc 301.16206, found 301.16237; HPLC
3ꢀThiomethylꢀestraꢀ1,3,5(10)ꢀtrienꢀ17βꢀol (7b)
To a solution of compound 7a (105 mg, 0.35 mmol) in MeOH/DCM 9:1 (20 mL) was
added under argon atmosphere and at 0 °C sodium borohydride (NaBH ) (12 eq.). The
4
mixture was then stirred at 0 °C under argon for 2 h. The reaction mixture was poured into
water and extracted with DCM. The organic phase was washed with water, dried with sodium
2 4
sulfate (Na SO ) and evaporated under reduced pressure. The crude compound was purified
by flash chromatography with hexanes/EtOAc (8:2) as eluent to give compound 7b as a white
1
amorphous solid (84 mg, 79%). IR (KBr) υ: 3379 (OH); H NMR (CDCl ) δ: 0.78 (s, CH ꢀ
3
3
1
1
8
8), 1.15ꢀ2.35 (m, residual CH and CH
2
), 2.46 (SCH
3 2
), 2.84 (m, CH ꢀ6), 3.73 (t, J = 8.4 Hz,
7αꢀCH), 7.00 (d, J = 1.8 Hz, CHꢀ4), 7.06 (dd, J
1
= 8.2 Hz, J2 = 2.1 Hz, CHꢀ2), 7.22 (d, J =
13
.2 Hz, CHꢀ1); C NMR (CDCl
3
) δ: 11.0, 16.2, 23.1, 26.1, 27.1, 29.5, 30.6, 36.7, 38.6, 43.2,
+
44.1, 50.1, 81.9, 124.5, 125.9, 127.5, 134.9, 137.4, 137.6; HRMS for C H OS [M + H] :
19
27
calc 303.17771, found 303.17815; HPLC purity: 95.7%.
ꢀ(Pyridinꢀ3ꢀylmethanethio)ꢀestraꢀ1,3,5(10)ꢀtrieneꢀ17ꢀone (8a)
To a solution of 3ꢀthioestrone (300 mg, 1.05 mmol), Cs CO
tetrabutylammonium iodide (TBAI) (416 mg, 1.12 mmol) in ACN (15 mL) was added 3ꢀ
3
2
3
(1.37 g, 4.2 mmol) and
29
ACS Paragon Plus Environment