Structure-Based Design and Synthesis of New Estrane-Pyridine Derivatives as Cytochrome P450 (CYP) 1B1 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00265

Inhibition of cytochrome P450 (CYP) 1B1 is a promising therapeutic strategy, as such an inhibitor could modulate the bioactivation of procarcinogens while reducing drug resistance. Based on docking studies, the synthesis of 12 estra-1,3,5(10)-triene derivatives containing a pyridin-3-/4-yl moiety at position C2, C3, or C4 was performed, and we measured their inhibitory activity on CYP1B1 using the ethoxyresorufin-O-deethylase (EROD) assay. The position of the nitrogen atom in the aromatic ring has little influence on their inhibition potency, but compounds with a pyridinyl at C2 of the steroid nucleus are more potent CYP1B1 inhibitors than those with a pyridinyl at C3 or C4. Estradiol derivatives (OH at C17β) are also 10-fold more potent inhibitors than estrone derivatives (carbonyl at C17). Thus, 2-(pyridin-3-yl)-estradiol (4a) is the best CYP1B1 inhibitor (IC₅₀ = 0.011 μM) from this series of compounds, and the best steroid inhibitor reported until now. It is also 7.5-fold more potent than the well-known nonsteroidal CYP1B1 inhibitor α-naphthoflavone (IC₅₀ = 0.083 μM).

Full text of DOI:10.1021/acsmedchemlett.7b00265

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Letter
Structure-Based Design and Synthesis of New Estrane-
Pyridine Derivatives as Cytochrome P450 (CYP) 1B1 Inhibitors
Raphaël Dutour, Francisco Cortés-Benítez, Jenny Roy, and Donald Poirier
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00265 • Publication Date (Web): 11 Oct 2017
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Structure-Based Design and Synthesis of New Estrane-Pyridine
Derivatives as Cytochrome P450 (CYP) 1B1 Inhibitors
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Raphaël Dutour,†,‡ Francisco Cortés-Benítez,†,# Jenny Roy,† and Donald Poirier *,†,‡
† Laboratory of Medicinal Chemistry, Endocrinology-Nephrology Unit, CHU de Québec – Research Center, Québec, Qc, Canada
‡ Department of Molecular Medicine, Faculty of Medicine, Université Laval, Québec, Qc, Canada
# Department of Pharmacy, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico
KEYWORD: Steroid, estrane, enzyme inhibitor, cytochrome P450, CYP1B1, molecular modelling
ABSTRACT: Inhibition of cytochrome P450 (CYP) 1B1 is a promising therapeutic strategy, as such an inhibitor could
modulate the bioactivation of procarcinogens while reducing drug resistance. Based on docking studies, the synthesis of
12 estra-1,3,5(10)-triene derivatives containing a pyridin-3-/4-yl moiety at position C2, 3 or 4 was performed and we meas-
ured their inhibitory activity on CYP1B1 using the ethoxyresorufin-O-deethylase (EROD) assay. The position of the nitro-
gen atom in the aromatic ring has little influence on their inhibition potency, but compounds with a pyridinyl at C2 of the
steroid nucleus are more potent CYP1B1 inhibitors than those with a pyridinyl at C3 or 4. Estradiol derivatives (OH at
C17β) are also 10-fold more potent inhibitors than estrone derivatives (carbonyl at C17). Thus, 2-(pyridin-3-yl)-estradiol
(4a) is the best CYP1B1 inhibitor (IC50 = 0.011 µM) from this series of compounds, and the best steroid inhibitor reported
until now. It is also 7.5-fold more potent than the well-known non-steroidal CYP1B1 inhibitor α-naphthoflavone (IC50 =
0.083 µM).
Introduction
depurinating adducts.12 In addition, it has been observed
that CYP1B1 is overexpressed in a wide variety of human
cancers (breast, lung, colon, esophagus, skin, testis,
lymph node and brain), but not in healthy tissue.13 Conse-
quently, CYP1B1 can also be a good marker for the preven-
tion of certain cancers. Finally, it should also be empha-
sized that CYP1B1 is involved in the metabolism of certain
anticancer agents, such as docetaxel, leading to drug-
resistance associated with the overexpression of CYP1B1.14-
16 Thus, a CYP1B1 inhibitor could be useful within a multi-
therapy context. Based on these observations, the inhibi-
tion of CYP1B1 constitutes a promising therapeutic strate-
gy, because it would enable a biological action at 3 dis-
tinct levels: 1) by inhibiting the formation of 4-hydroxy-
E2, 2) by inhibiting the bioactivation of procarcinogens,
and 3) by reducing drug-resistance.15
Cytochromes P450 (CYPs) form a large family of hemo-
proteins involved in many reduction and oxidation reac-
tions on both endogenic and xenobiotic compounds of
different sizes. Eighteen (18) CYP gene families, including
over 50 enzymes, are found in humans.1,2 The CYP1 family
is comprised of 3 members: CYP1A1, CYP1A2, and CYP1B1.
The latter is mainly expressed in extrahepatic mesoder-
mal cells, including steroidogenic tissues (ovaries, testes
and adrenal glands) and in steroid-responsive tissues
(breast, uterus and prostate).3 CYP1 enzymes have been
extensively studied because they are involved in the con-
version of many polycyclic aromatic hydrocarbons
(PAHs), such as benzo[α]pyrene, into carcinogens.4,5
Moreover, it should be noted that CYP1 enzymes are in-
volved in the modulation of pro-inflammatory and in-
flammatory pathways through the metabolism of leuko-
trienes and eicosanoids.6 These enzymes can also act as
tumor suppressors via the metabolism of some flavonoid-
and stilbene-type compounds.7-9
Over the past years, several CYP1 inhibitors have been
identified within different families, including flavonoids,
trans-stilbenes, coumarins, alkaloids and anthraquinone
derivatives, 1,17-19 but in the steroid family, only 16α-fluoro-
5-androsten-17-one and three methoxyestradiol deriva-
tives were reported as weak CYP1B1 inhibitors.20,21 This is
surprising, considering that C18-steroid E2 and its oxi-
dized form at position 17, estrone (E1), are both substrates
of several CYPs, including CYP1B1.11
Among these 3 CYP1 enzymes, CYP1B1 represents an in-
teresting therapeutic target, notably because it is involved
in the 4-hydroxylation of 17β-estradiol (E2) (Figure 1A),
whereas CYP1A1 and CYP1A2 mainly catalyse the 2-
hydroxylation of E2.10,11 Unlike 2-hydroxy-E2 and its oxida-
tion product E2-2,3-quinone, 4-hydroxy-E2 can be oxi-
dized into E2-3,4-quinone, a mutagenic compound which
can bind DNA covalently, leading to the formation of
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between a pyridine ring and the heme of CYP1B1 could be
achieved by adding a pyridine to the A-ring of E1 or E2.
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To address this idea, we performed docking simulations
using GOLD-5.4 software29,30 and the 3D structure of
CYP1B1 (PDB ID: 3PM0). The molecules screened in this
study were ANF for comparison purposes, and those rep-
resented in Figure 1B.
This screening provided a GOLD score (GS) of 69.8 and
a ChemPLP fitness score (CFS) of 95.5 for the best docked
conformation of ANF (Table 1). The 2-substituted com-
pounds, such as 3a (GS = 60.6 and CFS = 81.2), fit better
into the binding site of CYP1B1 than the corresponding 3-
substituted compound 6a (GS = 58.8 and CFS = 49.9) and
4-substituted compound 11a (GS = 45.6 and CFS = 76.0).
In fact, the pyridine ring of 3a interacts with the heme
group (Figure 2A) through a nitrogen-iron bond. Moreo-
ver, the nitrogen atom of 3a is closer to the iron than the
nitrogen atom of 6a (2.4 Å and 3.2 Å, respectively, Fig-
ures 2A and 2B). In addition, we found that the pyridinyl
moiety promotes an ideal orientation to perform pi-
stacking interactions between the A-ring of the steroid
and the side chain of Phe134. On the contrary, the struc-
ture of 11a is inverted when compared to 3a and 6a (Fig-
ure 2C). Thus, this orientation, suggesting an interaction
of the carbonyl at C17 with iron, significantly decreases
the interactions into the binding site. On the other hand,
4a (Figure 2D), which is an E2 derivative having a pyri-
din-3-yl at C2, presented the best GS and CFS for this
series (62.6 and 86.0, respectively).
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Figure 1. The conversion of estrogenic C18-steroid hormones by
CYP1B1 (A) and the structures of the newly synthesized CYP1B1
inhibitors (B). Partial numbering of carbons (left structure) and
steroid (A-D) ring identification (right structure) are reported.
Our research group has expertise in the development of
enzyme inhibitors of steroidogenesis having a steroid
nucleus (C18-, C19- or C21-steroids).22-25 In our previous
study, we reported the results obtained from the screen-
ing of 90 steroids and steroid derivatives on the CYP1B1
using the ethoxyresorufin-O-deethylase (EROD) assay.26
Among the molecules tested, we identified thioestrone
(IC50 = 3.4 μM) as an inhibitor of CYP1B1.26 Furthermore,
molecular modeling studies have shown that the 3-SH
group of this steroid is closer (3.36 Å) to the iron atom of
the heme system of CYP1B1 than the 3-OH of E1 and E2.
Based on these observations, we considered introducing
a chemical group on the A-ring of a C18-steroid (estra-
1,3,5(10)-triene) which could be able to interact with the
heme of CYP1B1. Pyridine, an aromatic ring containing a
nitrogen atom, is known to generate interactions with the
iron of heme systems.27 We therefore synthesized 12 ster-
oid derivatives containing a pyridin-3-/4-yl moiety at
position C2, 3 or 4 of E1 and E2 (Figure 1B). Their inhibi-
tory activity against CYP1B1 and docking simulations have
also been reported.
Results and discussion
Docking simulations and rational inhibitor design
The α-naphthoflavone (ANF) is one of the best CYP1B1
inhibitors reported in literature, and this flavonoid binds
the CYP1B1 catalytic site.28 The crystal structure of CYP1B1
(PDB ID: 3PM0) is also known, and it was used in our
design of new steroidal CYP1B1 inhibitors. We first divid-
ed the fused tricyclic core of ANF as three mimetic parts
for the A/B/C-rings of the steroidal scaffold. Furthermore,
in the co-crystallized structure of ANF, we observed that
the phenyl moiety at C2 was oriented towards the por-
phyrin and ferrous ion of the heme group. Thus, we be-
lieve that replacing this phenyl ring by a pyridine moiety
could generate a favorable interaction with the heme
group by means of a nitrogen-iron bond. Additionally, in
our previous docking studies using E1 and E2 (Supp. Info.,
Figure A), we observed that the A-ring was oriented to-
wards the heme group. However, it was less close when
compared to the phenyl moiety at C2 of ANF. From these
observations, we hypothesized that a desired interaction
Figure 2. Binding mode of 3a (A), 6a (B), 11a (C), and 4a (D) docked
in CYP1B1. Black sticks represent the heme group, whereas the iron
atom is highlighted as a red sphere. Images were produced using the
UCSF Chimera package.30
The binding mode for this compound showed the same
orientation and interactions as 3a. These include a nitro-
gen-iron bond with the heme as well as pi-stacking inter-
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6a and 6b were obtained by a Suzuki coupling of 5 with 3-
action between the A-ring and Phe134. Additionally, the
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steroidal core of 3a and 4a interacts by means of hydro-
phobic contacts with Phe268, Ala133, and Phe134. Never-
theless, 4a produced an H-bond between the 17β-OH
group and the carbonyl group from the side chain of
Asn228. This extra interaction is also performed by the E2
derivatives 4b, 7a as well as 7b (data not shown) and is
the main reason why we found better GS and CFS values
than 3b, 6a, and 6b. Hence, the E2 derivatives with a
pyridine ring linked to C2 could induce a significant in-
hibitory effect on CYP1B1.
or 4-pyridine boronic acid under the same reaction condi-
tions used for 2a and 2b. Compounds 6a and 6b were
then reduced with NaBH4 to give compounds 7a and 7b.
Scheme 2a
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Chemistry
To confirm our docking results, we performed the syn-
thesis of 12 E1 and E2 derivatives bearing a pyridin-3-/4-yl
moiety in C2, 3 or 4 of the estra-1,3,5(10)-triene (Figure
1B). A different synthesis pathway was used to introduce
the pyridine ring at each steroid position, and the final
step is the reduction of the C17 ketone into a secondary
alcohol. Compounds 3a, 3b, 4a, and 4b bearing a pyridi-
nyl moiety at C2 were obtained in two or three steps from
1 (Scheme 1). Compound 1 was previously obtained from
E1 in two steps: 1) 2-iodo-E1 was prepared via a selective
halogenation of E1 with mercuric acetate and iodine in
acetic acid31 and 2) the 3-OH was protected by a reaction
with the methoxymethyl (MOM) chloride in the presence
of Cs2CO3. Compounds 2a and 2b were obtained by a
Suzuki coupling between 1 and 3- or 4-pyridine boronic
acid in the presence of K3PO4 and the catalyst
Pd(dppf)Cl2. This reaction was performed in DMF under
microwaves (MW). Then, the MOM ether was hydrolysed
with HCl to form 3a and 3b. Finally, 4a and 4b were syn-
thesized from 3a and 3b through a reduction of the ke-
tone with sodium NaBH4.
aReagents and conditions: (a) Tf2O, 2,6-lutidine, DMAP, DCM, 0°C to
rt; (b) 3- or 4-pyridine boronic acid, Pd(dppf)Cl2, K3PO4, DMF, MW,
120°C, 2-3 h; (c) NaBH4, MeOH/DCM (9:1), 0°C, 2-3 h.
Steroid derivatives with a pyridinyl at C4 were synthe-
sized from 4-bromo-E1 (8) in three steps for the oxidized
forms 11a and 11b or in four steps for the reduced forms
12a and 12b (Scheme 3). Compound 8 was obtained from
E1 by a bromination with Br2 in the presence of powdered
iron, acetic acid, and water, as previously reported by
Slaunwhite and Neely.33 The MOM derivative 9 was
formed by the protection of the 3-OH of 8 with MOM-Cl
and Ce2CO3 in refluxing ACN. Thereafter, 10a and 10b
were obtained by a Suzuki coupling of 9 with 3- or 4-
pyridine boronic acid under different conditions than
those previously reported for C2 and 3. Indeed, due to the
lower reactivity of the bromine atom compared to the
iodine, the methodology used for the synthesis of com-
pounds with the pyridinyl at C2 or 3 was not efficient for
the coupling at C4. After investigation, we found alterna-
tive conditions for the Suzuki coupling of boronic acids
with aromatic compounds bearing a bromine atom. In
this methodology, Pd(PPh3)4 was used as the catalyst,
aqueous K2CO3 (2M) as the base, and the reaction was
carried out in a mixture of toluene and ethanol. Subse-
quent deprotection of the MOM ether with HCl led to the
formation of 11a and 11b. Compounds 12a and 12b were
next synthesized through a reduction of 11a and 11b with
NaBH4.
Scheme 1a
Scheme 3a
aReagents and conditions: (a) Hg(OAc)2, I2, AcOH, THF, rt, 2 h; (b)
MOM-Cl, Cs2CO3, ACN, reflux, 2-3 h; (c) 3- or 4-pyridine boronic
acid, Pd(dppf)Cl2, K3PO4, DMF, MW, 120°C, 2-3 h; (d) 10% HCl aq. in
MeOH (1:9), 50°C, overnight; (e) NaBH4, MeOH/DCM (9:1), 0°C, 2-3
h.
Steroid derivatives with a pyridinyl at C3 were obtained
from 5 in a single step for the oxidized forms 6a and 6b or
in two steps for the reduced forms 7a and 7b (Scheme 2).
A classical reaction between E1 and triflic anhydride with
DMAP as base gave 5 in a high yield of 90%.32 Compounds
aReagents and conditions: (a) Br2, 80% aq. acetic acid, Fe, 20°C; (b)
MOM-Cl, Cs2CO3, ACN, reflux, 2-3 h; (c) 3- or 4-pyridine boronic
acid, Pd(PPh3)4, toluene, EtOH, K2CO3 aq. (2M), reflux, overnight;
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a
(d) 10% HCl aq. in MeOH (1:9), 50°C, overnight; (e) NaBH4,
MeOH/DCM (9:1), 0°C, 2-3 h.
E1: estrone; E2: 17β-estradiol; T-E1: thioestrone; ANF: α-
b
naphthoflavone.
CFS: ChemPLP fitness score; GS: Gold score.
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cInhibition (% and IC50) of the transformation of resorufin ethyl
ether into resorufin by human CYP1B1 in presence of NADPH in
triplicate (±SD). See Supp. Info. (Figure B) for the inhibition curves
of 3a, 3b, 4a, 4b, and ANF.
The chemical reactions described above were generally
complete, but it is important to mention that a protection
of the 3-OH with a MOM group is necessary to subse-
quently perform the Suzuki coupling. In fact, this protect-
ing group promotes the formation of palladium complex-
es during this reaction, thus optimizing the coupling of
steroid derivatives 1 and 9 with pyridine boronic acid. In
this context, it should be noted that Ivanov et al. worked
on the optimization of the Suzuki-Miyaura reaction con-
ditions for the synthesis of 2-aryl-E1 and 2,4-diaryl-E1 by
using different ligands, and notably sterically emcum-
bered biaryl ligands.34 We were able to obtain E1 deriva-
tives with a pyridinyl at C2 in good yields (approximately
70-80%) under conventional Suzuki coupling reaction
conditions through the introduction of the MOM group
at C3.
The results of the EROD assay clearly highlight 4a and
4b, which have shown a very close inhibitory activity
against CYP1B1 (85.4 ± 0.3 and 87.4 ± 0.9%, respectively)
to that of ANF (94.0 ± 3.2%) at 0.3 μM. Compounds 4a
and 4b bear a pyridin-3-/4-yl, respectively, at C2 of the
steroid backbone. We also note that 3a and 3b, which are
distinguished from 4a and 4b only by the presence of a
ketone instead of an alcohol at C17, have a lower inhibito-
ry activity than their reduced forms. E2 derivatives appear
to be better inhibitors than their oxidized forms in most
cases, but the difference is particulary pronounced for 3a-
b and 4a-b. Compounds 6a-b and 7a-b, with a pyridinyl
at C3 show a weaker activity than their counterparts with
the pyridinyl in C2. Except 6b, which have the lowest
activity among the 12 synthesized compounds, these de-
rivatives at C3 present a similar inhibitory potency (~
20%). Finally, the C4-steroid derivatives 11a-b and 12a-b
have an inhibitory activity similar to that of 6a and 7a-b,
with the pyridinyl at C3. Compounds 12a-b have a slightly
higher inhibitory activity than those of the oxidized hom-
ologues 11a-b. Thioestrone is clearly a lower inhibitor in
comparison with these pyridine derivatives. Otherwise,
both substrates E1 and E2 showed a very weak inhibitory
potency towards CYP1B1 (~ 15% inhibition), thus demon-
strating the role of the pyridine ring.
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Structure-activity relationships against CYP1B1
The inhibitory activity of synthesized compounds 3a-b,
4a-b, 6a-b, 7a-b, 11a-b, and 12a-b against recombinant
human CYP1B1 enzyme was evaluated using the standard
EROD assay, which is widely used to assess CYP1 activity
(Table 1). In this assay performed in the presence of
NADPH as cofactor, the transformation of resorufin ethyl
ether, used as an enzyme substrate, into resorufin, was
measured by recording the fluorescence. ANF, a potent
CYP1B1 inhibitor,1 was used as the reference for this assay.
As the best steroidal CYP1B1 inhibitor,26 thioestrone was
included in the protocol to compare its activity with those
of our new steroid derivatives. The activity of E1 and E2,
two steroidal substrates of CYP1B1, was also evaluated.
From screening study results, we selected four steroid
derivatives with a pyridine ring at C2 as the best CYP1B1
inhibitors. From their inhibition curves, we determined
their IC50 values (Table 1), which allow a better compari-
son between each inhibitor.
Table 1. Docking Scores and Inhibition of CYP1B1
Activity by a Series of E1 and E2 Derivatives
Considering these results, the most important point is
that steroid derivatives with a pyridinyl at C2 (3a-b and
4a-b) are the best inhibitors of CYP1B1 activity among the
12 steroids bearing a pyridine moiety. This is quite inter-
esting, and confirms the docking results showing the best
CFS and GS when the pyridine ring was located at C2,
while the introduction of the pyridinyl in C3 or C4
seemed to lessen the affinity of these compounds for the
enzyme. Also note that E2 derivatives 4a-b are better
CYP1B1 inhibitors than their oxidized E1 homologues 3a-b
confirming our docking results. Given that E1 and E2 have
shown the same inhibitory activity against CYP1B1, this
result suggests that the hydroxy function in C17 is in-
volved in the formation of a H-bond, probably with the
Asn228 residue into the catalytic site of CYP1B1 as sug-
gested by our docking simulations. Moreover, it should be
stressed that 4a-b appear to be very potent inhibitors of
CYP1B1. Indeed, they have shown better inhibitory activi-
ties than ANF, a potent and well-known CYP1B1 inhibitor.
Inhibition
Inhibition
Cpda
X
Y
Z
GSb
CFSb
(%)
IC50( µM)c
at 0.3 µMc
3a
3b
4a
=O
=O
17βOH
17βOH
=O
N
CH
N
CH
N
CH
N
CH
N
CH
N
CH
-
-
-
CH
N
CH
N
60.6
52.5
62.6
54.2
58.8
56.2
45.7
49.0
45.6
43.2
44.1
42.8
58.6
48.5
69.8
69.8
81.2
74.8
86.0
81.8
49.9
49.7
85.7
84.4
76.0
74.8
71.2
71.9
49.7
53.6
-
37.8 ± 1.5
21.3 ± 3.1
85.4 ± 0.3
87.4 ± 0.9
28.2 ± 2.5
6.1 ± 2.2
0.063
0.120
0.011
0.032
4b
6a
6b
7a
CH
N
CH
N
-
-
=O
17βOH
17βOH
=O
19.6 ± 1.7
19.5 ± 0.7
14.0 ± 1.8
16.9 ± 2.3
28.9 ± 1.1
20.9 ± 4.5
16.0 ± 4.0
12.4 ± 3.0
13.3 ± 3.3
94.0 ± 3.2
-
-
7b
11a
11b
12a
12b
E1
E2
T-E1
ANF
CH
N
CH
N
-
-
-
-
=O
17βOH
17βOH
-
-
-
-
-
-
-
-
-
-
Plasmatic concentration of 4a in rats
-
-
95.5
0.083
A preliminary assessment of the stability of these new
CYP1B1 inhibitors was performed by determination of the
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important for the other derivatives with a pyridinyl in C3
plasmatic concentration of 4a as a function of time after a
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2
3
4
5
6
7
8
subcutaneous (sc) injection in rats (Figure 3). The con-
centration of the corresponding oxidized compound 3a
was also measured, because the secondary alcohol of an
E2 derivative is known to be metabolized by the Phase-I
metabolism enzymes. Surprisingly, the ketonic form of
4a, compound 3a, was more concentrated in plasma than
the alcohol 4a, but the shape of their concentration
curves was the same. In fact, following a sc injection of
0.66 mg of 4a in propylene glycol (PG):DMSO (92:8), the
plasmatic concentrations of 4a and 3a rapidly increased
to 4.0 and 7.0 ng/mL (at 2 h), respectively, and gradually
decreased thereafter. Although the plasmatic concentra-
tions measured and AUC values calculated (12.8, 33.2 and
46.0 ng.h/mL for 4a, 3a, and 4a + 3a, respectively) are
relatively acceptable for E2-derivatives, these preliminary
results highlight that both 17β-OH and 3-OH of 4a could
be protected towards Phase-I and phase-II metabolism, in
order to increase their metabolic stability and, conse-
quently, to increase their plasmatic concentrations.
or C4, but they are poor CYP1B1 inhibitors.
The position of the nitrogen atom into the pyridine ring
doesn’t strongly alter the inhibitory potency. Indeed,
compounds with a pyridin-3-yl moiety (series a) almost
have the same inhibitory profile as their analogues with a
pyridin-4-yl moiety (series b). However, the measurement
of IC50 values of 3a-b and 4a-b showed that 3a and 4a,
with a pyridin-3-yl at C2, were 2- and 3-fold more potent
CYP1B1 inhibitors than their counteparts 3b and 4b, with
a pyridin-4-yl at C2.
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The interesting results we obtained open the door to
the development of a new generation of steroidal CYP1B1
inhibitors. In fact, based on these SAR results and addi-
tional docking studies, the synthesis of a large series of
new and diversified estrane derivatives is planned to re-
veal their inhibitory activity against CYP1B1. Modification
at C17 will also be tested to avoid the formation of a less
potent ketone derivative, which is the major metabolite
observed when the corresponding alcohol was injected in
rats.
ASSOCIATED CONTENT
Supporting Information
1) Docking methodology, 2) experimental procedures for the
synthesis, characterization, and NMR spectra of all com-
pounds, 3) enzymatic EROD assay, 4) plasmatic concentra-
tion assay, 5) Figure A, 6) Figure B, and 7) references for
Supporting Information.
The Supporting Information is available free of charge on the
ACS Publications website.
Figure 3. Plasmatic concentrations of 2-(pyridin-3-yl)-E2 (4a) and 2-
(pyridin-3-yl)-E1 (3a) after a sc injection of 4a (0.66 mg in 500 µL
PG:DMSO (92:8); 2.0 mg/kg) in rats. AUC: area under curve.
AUTHOR INFORMATION
Corresponding Author
* Tel: 1-418-654-2296. E-mail: d0nald.poirier@crchul.ulaval.ca
Conclusion
Author Contributions
In conclusion, we have successfully synthesized a series
of estra-1,3,5(10)-triene derivatives bearing a pyridinyl
linked to the steroid A-ring (position C2, 3 or 4) and a
carbonyl or a hydroxyl group on D-ring (C17). This study
constitutes an important advance in the field of CYP1B1
inhibitors, as we have identified the most potent CYP1B1
inhibitors with a steroid scaffold. Indeed, 4a and 4b have
shown a better (7.5 and 2.5-fold) inhibitory activity when
compared to ANF, a potent non-steroidal CYP1B1 inhibi-
tor.
All authors contributed to writing the manuscript and have
given approval to the final version of the manuscript.
Funding Sources
Fonds de recherche du Québec – Santé (FRQS) for their
financial support from the Strategic Program.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
A major point of consideration is that the position of
the pyridine ring plays a key role for the inhibitory activi-
ty of the synthesized steroid derivatives and the best in-
hibitors appear to be those with the pyridinyl at C2. No-
tably, 4a and 4b (IC50 = 0.011 and 0.032 µM, respectively)
are 6- and 4-fold more active inhibitors against CYP1B1
activity than their oxidized homologues in C17 (3a and
3b), thus reflecting that 17β-OH contributes to improve
the affinity of these compounds for the catalytic site of
CYP1B1. However, the difference of inhibitory activity
between the reduced form and the oxidized form is not so
Francisco Cortés Benítez thanks the National Autono-
mous University of Mexico and the National Council for
Sciences and Technology (CONACyT) for the fellowship
awarded. Careful reading of this manuscript by Mrs. Michel-
ine Harvey is also greatly acknowledged.
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