Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives

Article abstract of DOI:10.1080/14756366.2018.1452919

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-β-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhib

Full text of DOI:10.1080/14756366.2018.1452919

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Blocking oestradiol synthesis pathways with
potent and selective coumarin derivatives
Sanna Niinivehmas, Pekka A. Postila, Sanna Rauhamäki, Elangovan
Manivannan, Sami Kortet, Mira Ahinko, Pasi Huuskonen, Niina Nyberg, Pasi
Koskimies, Sakari Lätti, Elina Multamäki, Risto O. Juvonen, Hannu Raunio,
Markku Pasanen, Juhani Huuskonen & Olli T. Pentikäinen
To cite this article: Sanna Niinivehmas, Pekka A. Postila, Sanna Rauhamäki, Elangovan
Manivannan, Sami Kortet, Mira Ahinko, Pasi Huuskonen, Niina Nyberg, Pasi Koskimies, Sakari
Lätti, Elina Multamäki, Risto O. Juvonen, Hannu Raunio, Markku Pasanen, Juhani Huuskonen
& Olli T. Pentikäinen (2018) Blocking oestradiol synthesis pathways with potent and selective
coumarin derivatives, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 743-754, DOI:
10.1080/14756366.2018.1452919
To link to this article: https://doi.org/10.1080/14756366.2018.1452919
© 2018 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 743–754
https://doi.org/10.1080/14756366.2018.1452919
RESEARCH PAPER
Blocking oestradiol synthesis pathways with potent and selective coumarin
derivatives
a
a
a
e
ꢀ
ꢀ
€
Sanna Niinivehmas , Pekka A. Postila
, Sanna Rauhamaki
, Elangovan Manivannana,b, Sami Korteta,c,
a
d
d
a
a
d
€
€
Mira Ahinko , Pasi Huuskonen , Niina Nyberg , Pasi Koskimies , Sakari Latti , Elina Multamaki , Risto O. Juvonen ,
Hannu Raunio , Markku Pasanen , Juhani Huuskonen and Olli T. Pentikainen
d
d
c
a,f
€
b
aDepartment of Biological and Environmental Science and Nanoscience Center, University of Jyvaskyla, Jyvaskyla, Finland; School of Pharmacy,
c
d
Devi Ahilya University, Indore, India; Department of Chemistry and Nanoscience Center, University of Jyvaskyla, Jyvaskyla, Finland; School of
e
f
Pharmacy, University of Eastern Finland, Kuopio, Finland; Forendo Pharma Ltd, Turku, Finland; Institute of Biomedicine, University of Turku,
Turku, Finland
ABSTRACT
ARTICLE HISTORY
Received 12 January 2018
Revised 8 March 2018
Accepted 8 March 2018
A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking
oestradiol synthesis by 17-b-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase
pathway. Five analogues produced ꢁ62% HSD1 inhibition at 5 mM and, furthermore, three of them pro-
duced ꢁ68% inhibition at 1 mM. A docking-based structure-activity relationship analysis was done to deter-
mine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against
oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues
are only modestly active with 17-b-hydroxysteroid dehydrogenase 2 – a requirement for lowering effective
oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin
as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety
substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer
and endometriosis.
KEYWORDS
3-Phenylcoumarin;
17-b-hydroxysteroid
dehydrogenase 1 (HSD1);
3-imidazolecoumarin;
aromatase; structure-activity
relationship (SAR)
Introduction
E2 (Figure 1(A,B)). In contrary, 17-b-hydroxysteroid dehydrogenase
2 (HSD2 or 17-b-HSD2) promotes the oxidation of the C17-
hydroxyl group on E2 by donating Hþ to the cofactor to produce
E1. HSD1 overexpression is a strong signal for breast cancer – pre-
sent in ꢂ50% of breast tumours – and, furthermore, HSD2 is
Despite the recent advances made in early tumour detection, clin-
ical treatments and avoidance of menopausal hormone therapies,
breast cancer continues to be the most common invasive cancer,
and a second leading cause of cancer death for women1. Therefore,
potent and selective pharmaceutical agents are actively sought to
supplement and/or replace the often-invasive treatments and to
lower the medical costs for all breast cancer patients.
A clear majority of breast cancer tumours are oestrogen recep-
tor (ER) positive. The tumour growth is linked to high ER numbers
and/or their increased activity due to high 17-b-oestradiol (E2) lev-
els. Hence, the existing drugs generally aim to block the ER func-
tion in breast tissue or limit its function indirectly by lowering the
E2 production. The aromatase pathway produces E2 from andro-
gen hormones whereas the sulphatase pathway converts oestrone
sulphate (E1S) into oestrone (E1) and ultimately to E2. Although
the aromatase pathway (active in local E2 production) is in a lesser
role with most breast cancers2, widely used drugs, such as anas-
trozole focus on blocking it instead of the more prominent sulpha-
tase pathway.
known to have an inhibitory effect in the breast tumourigenesis3,4
.
HSD1 is also linked to other cancer types, such as gastric5 and cer-
vical cancer6, and, additionally, in endometriosis elevated E2 pro-
duction is promoted by increased HSD1 and, inversely, lowered
HSD2 expression7.
A vast number of steroidal (8–10; e.g. E2B in Figure 1(C)) and
non-steroidal (see e.g.11–13) compounds are known to inhibit the
HSD1 activity, but none of these promising leads has passed clin-
ical trials so far. There are also several X-ray crystal structures of
HSD1 in both ligand-free, substrate-, and inhibitor-bound states to
facilitate rational structure-based drug discovery. Here, 3-phenyl-
coumarin (or 3-arylcoumarin) is shown to be a suitable non-ster-
oidal scaffold for building small-molecule inhibitors targeting
HSD1 (Figure 2; Table 1).
Altogether, nine 3-phenylcoumarin analogues with varying cou-
marin and 3-phenyl ring substituents (R1–R6 positions; Figure 2)
were synthesised (Table 1). Five of the analogues produced ꢁ62%
HSD1 inhibition at 5 mM and, furthermore, three of them elicited
ꢁ68% inhibition even at 1 mM (estimated pIC50 ꢁ 6.2). The docking-
17-b-hydroxysteroid dehydrogenase 1 (HSD1 or 17-b-HSD1;
Figure 1(A)) has a crucial role in the final steps of E2 biosynthesis
via the sulphatase pathway. HSD1 homodimer reduces the C17-
keto group of E1 by acquiring a proton (Hþ) from the cofactor
nicotinamide adenine dinucleotide phosphate (NADPH) to produce based structure-activity relationship (SAR) analysis indicates that the
€
CONTACT Olli T. Pentikainen
olli.pentikainen@utu.fi
Department of Biological and Environmental Science and Nanoscience Center, University of Jyvaskyla,
Jyvaskyla, Finland
ꢀ
These authors contributed equally to this work.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

744
S. NIINIVEHMAS ET AL.
Figure 1. The ligand binding at the active site of 17-b-hydroxysteroid dehydrogenase 1. (A) Oestradiol (E2; orange backbone) and oxidised cofactor nicotinamide
adenine dinucleotide phosphate (NADP; blue) are shown as CPK models in complex with the HSD1 structure (grey cartoon; PDB: 1A27). (B) The H-bonding between E2
(ball-and-stick models with orange backbone) and the residues lining the active site (stick model with black backbone) are shown with magenta dotted lines. The sub-
strate oestrone (E1) acquires a proton (or Hþ) from NADPH, the reduced form of the cofactor, via the hydroxyl group of Tyr156 (E2 þ NADPþ þ ꢀ E1 þ NADPH), which
is H-bonding with the 17-keto group of the reaction product E2. (C) Inhibitor E2B (ball-and-stick model with orange backbone; PDB: 3HB5)25 binding at the HSD1 active
site blocks E2 binding (B vs. C). (D) The 3-phenyl and coumarin rings of the docked analogues (stick model with green backbone) align in a roughly similar manner
inside the active site as the steroid ring of E2B (stick model with orange backbone).
Figure 2. 2D structures of the coumarin derivatives. The 3-phenylcoumarin analogues 1–7 produce HSD1 inhibition at a varying degree, but 8 and 9 were found to be
inactive (Table 1). Compound 10 or 3-imidazolecoumarin inhibit aromatase instead of HSD1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
745
potent analogues mimic steroid binding (Figure 1(D)). A cross-
reactivity profile, covering HSD2, monoamine oxidases A (MAO-A)
and B (MAO-B), ER, cytochrome P450 1A2 (CYP1A2), and aromatase
(or CYP19A1), were built for each analogue. Importantly, the substitu-
tion of the 3-phenyl ring with the 3-imidazole ring in the coumarin
core, assures strong and selective aromatase inhibition.
In short, the coumarin-based compounds have potential for
lowering E2 levels needed in battle against diseases, such as breast
cancer or endometriosis by blocking either the aromatase pathway
or the sulphatase pathway.
Methods
Chemical procedure
All reactions were carried out using commercial materials and
reagents without further purification unless otherwise noted.
Reaction mixtures were heated by the CEM Discovery microwave
apparatus. All reactions were monitored by thin layer chromatog-
raphy (TLC) on silica gel plates. 1H NMR and 13C NMR data were
recorded on a Bruker Avance 400 MHz spectrometer or Bruker
Avance III 300 MHz spectrometer (Bruker, Billerica, MA). Chemical
shifts are expressed in parts per million values (ppm) and are des-
ignated as singlet (s), broad singlet (br s), doublet (d), double
doublet (dd), double double doublet (ddd), and triplet (t).
Coupling constants (J) are expressed as values in hertz (Hz). The
HRMS mass spectra were recorded using Micromass LCT ESI-TOF
equipment (Waters Corporation, Milford, MA). Elemental analyses
were done with Elementar Vario EL III elemental analyser
(Elementar-Straße 1, Langenselbold, Germany). The 3-phenylcou-
marin analogues were synthesised using Perkin–Oglialor condensa-
tion reaction. The method was developed from the earlier
published procedures and transferred to microwave reactor.
Experimental data for 7-hydroxy-3–(4-fluorophenyl)-2H-chro-
men-2-one (5; Figure 2), 7-hydroxy-3–(4-methoxyphenyl)-2H-chro-
men-2-one (8; Figure 2) and 7-hydroxy-3–(4-hydroxyphenyl)-2H-
chromen-2-one (9; Figure 2) have been published14. However, the
synthesis steps are detailed below for other derivatives studied
here (1, 2, 3, 4, 6, 7 and 10 Figure 2; Scheme 1). Of these 1–4
have also been synthesised earlier by others prior to this
study15–18. 2; Scheme 1).
A typical procedure (Scheme 1): A mixture of salicylaldehyde
derivative (2 mmol) and phenylacetic acid derivative (2.1 mmol),
acetic acid anhydride (0.6 ml), and triethylamine (0.36 ml) were
placed in a microwave reactor tube and this mixture was heated
at 100–170 ꢃC with microwave apparatus for 10–20 min. After cool-
ing, 2 ml of 10% NaHCO3 solution was added and the precipitate
was filtered, dried, and recrystallised from ETOH/H2O or acetone/
H2O mixture. The acetyl group(s) were removed by treating the
compound with MeOH/NaOH(aq) solution for 30–60 min at r.t. The
solution was acidified with HCl(aq,) and the precipitate was filtered
and recrystallised if needed.
Based on the elemental analysis and/or 1H-NMR the purity of
compounds was >95%.
8-hydroxy-3–(4-methoxyphenyl)-2H-chromen-2-one (1)15. In
the first step 8-acetoxy-3–(4-methoxyphenyl)-2H-chromen-2-one
was obtained. Yield 85%; 1H-NMR (400 MHz, d6-DMSO) d: 2.40 (s,
3H, CH3C(O)O-Ph), 3.80 (s, 3H (CH3O-Ph), 7.02 (d, 2H, J3 ¼ 8.1 Hz,
H-30, H-50), 7.37 (t, 1H, J3 ¼ 7.6 Hz, H-6), 7.43 (d, J3 ¼ 7.7 Hz, 1H,
H-7), 7.64–7.69 (m, 3H, H-5, H-20, H-60), 8.11 (s, 1H, H-4); 13 C-NMR
(100.6 MHz, d6-DMSO) d: 20.33, 55.20, 113.70, 120.85, 124.41,
124.65, 125.87, 126.50, 126.80, 129.82, 136.70, 138.88, 144.38,
158.83, 159.73 and 168.38. HRMS(ESI): calcd for C18H14O5Na1
[M þ Na]þ: 333.07389, found 333.07580. Elemental anal. for

746
S. NIINIVEHMAS ET AL.
O
128.31, 129.41, 131.45, 135.67, 139.82, 150.30, 151.66, 159.10
acetic
anhydride
R
O
OH
and169.22. In the second step, 6-chloro-3–(3-hydroxyphenyl)-2H-
R'
R
R'
+
H
1
chromen-2-one was obtained. Yield 80%; %; H-NMR (400 MHz, d6-
TEA,
mw 10 min
O
40
O
DMSO) d:), 6.84 (ddd, 1H, J3 ¼ 8.0 Hz, J4 ¼ 2.4 Hz, J ¼ 2.3 Hz, H-60),
OH
Scheme 1. The synthesis of 3-phenylcoumarin analogues and 3-imidazolecoumarin.
7.10–7.15 (m, 2H), 7.27 (t, 1H, J3 ¼ 7.9 Hz, H-50), 7.47 (d, 1H,
J3 ¼ 8.9 Hz, H-8), 7.65 (dd, 1H, J3 ¼ 8.3 Hz, J4 ¼ 2.6 Hz, H-7), 7.90 (d,
1H, J4 ¼ 2.5 Hz, H-5), 8.17 (s, 1H, H-4), 9.57 (s, 1H, Ph-OH); 13 C-NMR
(75.5 MHz, d6-DMSO) d: 115.43, 115.85, 117.79, 119.13, 120.84,
127.53, 127.99, 128.18, 129.26, 131.09, 135.44, 139.03, 151.50,
157.08 and 159.11. HRMS (ESI): Calcd for C15H9Cl1O3Na1 [M þ Na]þ:
295.01379, found 295.01380.
C18H14O5, calc. C% 69.67, H% 4.55, found C% 69.53, H% 4.55.
In the second step, 8-hydroxy-3–(4-methoxyphenyl)-2H-chromen-
2one was obtained. Yield 81%; 1H-NMR (400 MHz, d6-DMSO)
d: 3.80 (s, 6H (CH3Oꢄ), 7.01 (d, J3 ¼ 8.9 Hz, 2H, H-30,H-50), 7.08 (dd,
1H, J3 ¼ 7.0 Hz, J4 ¼ 2.6 Hz, H-7), 7.12–7.18 (m, 2H, H-5, H-6), 7.70
(d, 2H J3 ¼ 8.9 Hz, H-20,H-60), 8.11 (s, 1H, H-4), 10.19 (s, 1H, Ph-OH).
13 C-NMR (100.6 MHz, d6-DMSO) d: 55.21, 113.64, 117.64 118.39,
120.55, 124.45, 126.22, 126.91, 129.79, 139.52, 141.42, 144.26,
159.54 and159.76. HRMS(ESI)): calcd for C16H12O4Na1 [M þ Na]þ:
291.06333, found 291.06180. Elemental anal. for C16H12O4, calc. C%
71.26, H% 4.51, found C% 71.64, H% 4.51.
3–(3-fluoro-4-hydroxyphenyl)-7-methoxy-2H-chromen-2-one
(6). In the first step, 2-fluoro-4–(7-methoxy-2-oxo-2H-chromen-3-
yl)phenyl acetate was obtained. Yield 75%; 1H-NMR (400 MHz, d6-
DMSO) d: 2.35 (s, 3H, CH3C(O)O-Ph), 3.88 (s, 3H, CH3O-Ph), 6.99
(dd, 1H, J3 ¼ 8.6 Hz, J4 ¼ 2.4 Hz, H-6), 7.05 (d, 1H, J4 ¼ 2.4 Hz, H-8),
7.37 (t, 1H, J ¼ 8.3 Hz, H-60), 7.62 (d, J ¼ 8.5 Hz, 1H, H-50), 7.68 (d,
J ¼ 8.6 Hz, 1H, H-5), 7.74 (dd, JH-F ¼ 12.1 Hz, J4¼ 2.0 Hz, H-30), 8.31
(s, 1H, H-4); 13 C-NMR (100 MHz, d6-DMSO) d: 20.19, 55.97, 100.25,
112.79, 116.35 (d, JC–F ¼ 20 Hz), 121.02 (d, JC-F ¼ 1.9 Hz), 123.83,
124.79 (d, JC-F ¼ 3.2 Hz), 129.86, 134.24 (d, JC–F ¼ 7.7 Hz), 137.20 (d,
JC-F ¼ 13.1 Hz), 141.55, 153.00 (JC-F ¼ 246.1 Hz), 154.92, 159.65 and
162.69, 168.19. In the second step, 3–(3-fluoro-4-hydroxyphenyl)-7-
methoxy-2H-chromen-2-one was obtained. Yield 70%; 1H-NMR
(400 MHz, d6-DMSO) d: 3.87 (s, 3H, CH3O-Ph), 6.96–7.03 (m, 3H, H-
6, H-8, H-50), 7.41 (d, 1H, J3 ¼ 8.4, H-60), 7.57 (dd, 1H, JH-F ¼ 13.1 Hz,
J4 ¼ 2.2 Hz (H-H), 1H, H-20), 7.66 (d, 1H, J3 ¼ 8.4, H-5), 8.18 (s, 1H, H-
4), 10.09 (s, 1H, Ph-OH). 13 C-NMR (75.5 MHz, d6-DMSO) d: 55.91,
100.16, 112.61, 113.04, 115.95 (d, JC-F ¼ 20 Hz), 117.37 (d, JC-F
¼ 3.3 Hz), 121.78 (JC-F ¼ 2.0 Hz), 124.54 (d, JC-F ¼ 3.0 Hz), 126.08 (d,
JC-F ¼ 7.0 Hz), 129.49, 139.62, 145.0 (JC-F ¼ 13 Hz), 150.46 (d, JC-
6-hydroxy-3–(4-hydroxyphenyl)-2H-chromen-2-one (2)19. In
the first step 4–(6-acetoxy-2-oxo-2H-chromen-3-yl)phenyl acetate
was obtained. Yield 90%; 1H-NMR (300 MHz, d6-DMSO) d: 2.30 (s,
3H, CH3CO(O)-Ph), 2.31 (s, 3H, CH3CO(O)-Ph), 7.23 (d, 2H,
J3 ¼ 8.8 Hz, H-20, H-60), 7.40 (dd, J3 ¼ 8.9 Hz, J4 ¼ 2.7 Hz, 1H, H-7),
7.49 (d, 1H, J3 ¼ 8.9 Hz, H-8), 7.55 (d, 1H, J4 ¼ 2.6 Hz, H-5), 7.76 (d,
2H, J3 ¼ 8.8 Hz, H-30, H-50), 8.24 (s, 1H, H-4); 13 C-NMR (75.5 MHz,
d6-DMSO) d: 20.73, 20.82, 116.97, 119.90, 120.77, 121.67, 125.48,
126.67, 129.74, 131.95, 139.84, 146.43, 150.43, 150.76, 159.51,
169.10 and 169.22. In the second step, 6-hydroxy-3–(4-hydroxy-
phenyl)-2H-chromen-2-one was obtained. Yield 85%; 1H-NMR
(400 MHz, d6-DMSO) d: 6.83 (d, 2H, J3 ¼ 8.8 Hz, H-30, H-50), 6.99 (dd,
1H, J3 ¼ 8.8 Hz, J4 ¼ 2.9 Hz, H-7), 7.06 (d, 1H, J4 ¼ 2.8 Hz, H-5), 7.24
(d, 1H, J3 ¼ 8.9 Hz, H-8), 7.57 (d, 2H, J3 ¼ 8.7 Hz, H-20, H60), 8.04 (s,
1H, H-4); 13 C-NMR (75.5 MHz, d6-DMSO) d: 112.29, 115.00,116.59,
119.15, 120.24, 125.40, 126.71 129.86, 138.51, 146.03, 153.77,
157.90 and 160.13. HRMS(ESI)): calcd for C16H11F1O4Na1 [M þ Na]þ:
277.0477, found 277.0461.
F
¼ 240 Hz), 154.52, 159.87 and 162.19. HRMS (ESI): Calcd for
C16H11F1O4Na1 [M þ Na]þ: 309.0539, found 309.0553.
3–(3-fluoro-4-hydroxyphenyl)-6-methoxy-2H-chromen-2-one
(7). In the first step, 2-fluoro-4–(6-methoxy-2-oxo-2H-chromen-3-
yl)phenyl acetate was obtained. Yield 66%; 1H-NMR (400 MHz, d6-
DMSO) d: 2.33 (s, 3H, CH3C(O)O-Ph), 3.82 (s, 3H (CH3O-Ph), 7.23
(dd, 1H, J3 ¼ 9.0 Hz, J4 ¼ 3.0 Hz, H-7), 7.30 (d, 1H, J4 ¼ 3.0 Hz, H-5),
7.35 (d, 1H, J3 ¼ 9.2 Hz, H-8), 7.61 (d, 1H, J3 ¼ 8.5 Hz, H-50), 7.75 (dd,
1H, JH-F ¼ 12.0 Hz, J4 ¼ 1.7 Hz (H-H), 1H, H-20), 8.30 (s, 1H, H-4); 13 C-
NMR (100.6 MHz, d6-DMSO) d: 20.22, 55.69, 110.83, 116.67, 117.02,
119.66, 123.96, 125.10, 135.96, 141.18, 147.44, 151.78, 154.23,
155.70, 159.53 and 168.21. In the second step, 3–(3-fluoro-4-
hydroxyphenyl)-6-methoxy-2H-chromen-2-one was obtained. Yield
71%; 1H-NMR (400 MHz, d6-DMSO) d: 3.81 (s, 3H (CH3O-Ph), 7.02
(dd, 1H, J3 ¼ 9.2 Hz, H-60), 7.18–7.28 (m, 1H, H-5, H-7), 7.35 (d,
J3 ¼ 9.0 Hz, H-8), 7.42 (d, 1H, J3 ¼ 8.4 Hz, H-50), 7.57 (dd, 1H,
JH-F ¼ 13.0 Hz, J4 ¼ 2.2 Hz (H-H), 1H, H-20), 8.17 (s, 1H, H-4), 10.19 (s,
1H, Ph-OH); 13 C-NMR (100.6 MHz, d6-DMSO) d: 55.66, 110.59,
116.67, 117.02, 119.66, 123.96, 125.10, 135.96, 141.18, 147.44,
151.78, 154.23, 155.70, 159.53 and 168.21. HRMS (ESI): Calcd for
C16H11F1O4Na1 [M þ Na]þ: 309.0539, found 309.0521.
3–(3-hydroxyphenyl)-2H-chromen-2-one (3)20. In the first
step, 3–(2-oxo-2H-chromen-3-yl)phenyl acetate was obtained. Yield
87%; 1H-NMR (400 MHz, d6-DMSO) d: 2.30 (s, 3H, CH3C(O)O-Ph),
40
7.20 (ddd, 1H, J3 ¼ 9.0 Hz, J4 ¼ 2.2 Hz, J ¼ 2.3 Hz, H-60), 7.39 (t, 1H,
J3 ¼ 7.6 Hz, H-50), 7.44 (d(broad), 1H, J3 ¼ 8.3 Hz, H-40), 7.49–7.53 (m,
2H, H-6, H-20), 7.62–7.66 (m, 2H, H-7, H-8) 7.79 (dd, 1H, J3 ¼ 8.7 Hz,
J4 ¼ 1.5 Hz, H-5), 8.32 (s, 1H, H-4); 13 C-NMR (100 MHz, d6-DMSO)
d: 20.86, 115.90, 119.38, 121.81, 122.17, 124.68, 125.69, 125.90,
128.81, 129.31, 131.98, 135.99, 141.13, 150.30, 153.02, 159.51 and
169.23. In the second step, 3–(3-hydroxyphenyl)-2H-chromen-2-
one was obtained. Yield 74%; H-NMR (300 MHz, d6-DMSO) d: 6.83
1
40
(ddd, 1H, J3 ¼ 8.1 Hz, J4 ¼ 2.2 Hz, J ¼ 2.4 Hz, H-40), 7.11–7.18 (m,
2H, H-20, H-60), 7.26 (t, 1H, J3 ¼ 7.9, H-50), 7.37 (ddd, 1H, J3 ¼ 7.6 Hz,
0
J4 ¼ 1.1 Hz, J4 ¼ 1.1 Hz, H-6), 7.42 (d, J3 ¼ 8.3 Hz, H-8), 7.61 (ddd,
J3 ¼ 7.3 Hz, J4 ¼ 1.6 Hz, J4 ¼ 2.6 Hz H-7), 7.83 (dd, 1H, J ¼ 8.7 Hz,
3
0
J4 ¼ 1.5 Hz, H-5), 8.20 (s, 1H, H-4), 9.54 (s, 1H,Ph-OH); 13 C-NMR
(75.5 MHz, d6-DMSO) d: 115.45, 115.59, 115.76, 119.13, 119.43,
124.50, 126.86, 128.60, 129.20, 131.60, 135.79, 140.32, 152.87,
157.06, and 159.54. HRMS (ESI): Calcd for C15H10O4Na1 [M þ Na]þ:
261.05276, found 261.04980.
3–(1H-imidazol-1-yl)-2H-chromen-2-one (10). Yield: 39% light
1
brown solid; Rf ¼0.18 (EtOAc); H-NMR (300 MHz, d6-DMSO) d: 7.10
(br s, 1H, H-40), 7.44 (apparent td, J3 ¼ 7.5 Hz, J4 ¼1.0 Hz, 1H, H-6),
7.51 (d, J3¼8.3 Hz, 1H, H-8), 7.64–7.70 (m, two overlapping signals,
2H, H-7 and H-50), 7.77 (dd, J3¼7.7 Hz, J4 ¼1.5 Hz, 1H, H-5), 8.16 (br
s, 1H, H-20), 8.34 (s, 1H, H-4). 13 C-NMR (75 MHz, d6-DMSO)
d: 116.06 (C-H8), 118.51 (H5-C-C-C-H4), 119.57 (C-H50), 123.37
(N-C-C ¼ O), 125.09 (C-H6), 128.63 (C-H40), 128.80 (C-H5), 131.87 (C-
H7), 132.97 (C-H4), 137.12 (N-C(-H20)¼N), 151.78 (H8-C-C-O), 156.83
(C¼O). IR (KBr): 1727, 1708, 1630, 1608, 1486, 1318, 1083 and 760.
6-chloro-3–(3-hydroxyphenyl)-2H-chromen-2-one (4)21. In
the first step, 3–(6-chloro-2-oxo-2H-chromen-3-yl)phenyl acetate
was obtained. Yield 85%; 1H-NMR (400 MHz, d6-DMSO) d: 2.30 (s,
3H, CH3C(O)O-Ph), 7.22 (ddd, 1H, J3 ¼ 8.0 Hz, J4 ¼ 2.2 Hz,
40
J ¼ 2.3 Hz, H-60), 7.48–7.52 (m, 3H, H-8, H-20, H-50), 7.62 (m, 1H, H-
40), 7.67 (dd, 1H, J3 ¼ 8.9 Hz, J4 ¼ 2.6 Hz, H-7), 7.88 (d, 1H,
J4 ¼ 2.6 Hz, H-5), 8.27 (s, 1H, H-4).); 13 C-NMR (100 MHz, d6-DMSO) d: ESI-MS: m/z (rel. abund. %): calculated for (M þ Naþ) ¼ 235.0478,
20.85, 117.96, 120.78, 121.85, 122.47, 125.93, 126.88, 127.70, measured 235.0476, D ¼ 0.2 mDa. Elemental analysis for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
747
C12H8N2O2: calc. C% 67.92, H% 3.80, N% 13.20, found C% 67.49, immediately followed and the plates were read 300 times every 15 s
H% 3.72 and N% 13.13. Mp. 180–182 ꢃC.
using 1 s exposure time. The assay should produce absorbance
change of ꢂ0.3523. The more active MAO-A produced over 0.5
change in absorbance reaching the assay maximum in 30 min with
25 mg of protein (enzymatic activity 5.25 units) per well while MAO-B
17-b-Hydroxysteroid dehydrogenase 1 and 2
produced the expected 0.35 change in absorbance with 50mg of
The inhibition was determined by HPLC using recombinant human
HSD1 and HSD2 proteins as described in a prior study10. In short, protein (enzymatic activity 3.2 units) per well and reached the assay
recombinant human HSD1 and HSD2 were produced in Sf9-insect
cells. The assay was performed in a final volume of 0.2 ml buffer
(20 mM KH2PO4, 1 mM EDTA, pH 7.4) containing 0.1 mg/ml protein,
1 mM cofactor (NADPH for HSD1, NAD for HSD2), 30 nM substrate
oestrone or oestradiol, 800,000 cpm/ml of tritium labelled oestrone
([3H]-E1) or oestradiol ([3H]-E2), and inhibitors concentrations in
the range of 0.1–5.0 mM. Triplicate samples were incubated for
25 min at the room temperature. After incubation, the reaction
was stopped by addition of 20 ml 10% trichloroacetic acid per
sample. After incubation the substrate and the product of enzym-
atic conversion [3H]-E1 and [3H]-E2 were separated and quantified
by HPLC (Alliance 2790, Waters, Milford, MA) connected to an
online counter (Packard Flow Scintillation Analyser; Perkin Elmer
Inc., Waltham, CA). The ratio of [3H]-E1 converted to [3H]-E2 or
vice versa determines the conversion percentage of the samples.
Inhibition was measured in three concentrations (100 nM, 1, and
5 mM) in order to follow the progression of inhibition efficiencies.
Inhibition efficiencies of the tested inhibitors were calculated by
comparing the conversion percentages of the samples including
inhibitors with those of conversion controls (without inhibitors).
The pIC50 average values and their standard errors were estimated
from three measurements at 1 mM.
maximum in 2 h. These protein concentrations were selected to be
used to analyse the molecules 1–9. The analysis conditions followed
the above-described assay protocol23 and the activity of tested mol-
ecules was measured at 100 mM for MAO-A and at 10 mM for MAO-B.
The analysis was performed as single point measurements and the
signal was read by the same instrument at the expected assay max-
imum indicated by the activity measurements, at 30 min for MAO-A
and at 2 h for MAO-B, respectively. Clorgyline was used as MAO-A
and pargyline as MAO-B inhibitor control. Both of the control inhibi-
tors provided 100% inhibition at the assay concentration of the test
molecules. In addition, pIC50 values were determined for MAO-B
inhibition using duplicated dilution series and the pIC50 value calcu-
lated for MAO-B inhibition by pargyline was 6.21. The observed
activity was calculated as inhibition percentage (Table 1). The pIC50
values were calculated with GraphPad Prism version 5.03 (GraphPad
Software Inc., San Diego, CA).
Oestrogen receptor
The pIC50 values of the molecules (Table 1) were measured using
green PolarScreenTM ER Alpha Competitor Assay (Life Technologies,
Carlsbad, CA) kit, following the protocol provided by the manufac-
turer as previously described14. The final concentration of the mol-
ecules ranged between 0.0007 and 10,000 nM in the prepared
dilution series. The molecules were combined with 25 nM ERa and
4.5 nM fluormone in the assay buffer and placed on black low vol-
ume 384-well assay plate with NBS surface (Corning Inc., Corning,
NY). After mixing the assay plate, it was incubated for 2 h at the
room temperature. The fluorescence polarisation was then meas-
ured using excitation wave length 485 and emission wave length
Aromatase
Aromatase (CYP19A1) activity was measured as described previ-
ously22 by using human placental microsomes and 50 nM [3H]-
androstenedione as a substrate and inhibitor concentrations in the
range of 60–1000 nM. Aromatase activities were measured as
released [3H]-H2O in Optiphase Hisafe 2 scintillation liquid (Perkin
Elmer, Waltham, MA) with a Wallac 1450 MicroBeta Trilux scintilla-
tion counter (Perkin Elmer, Waltham, MA). As a positive control for
aromatase inhibition, 1 mM finrozole (generous gift from Olavi
Pelkonen, University of Oulu, Finland) was used.
R
535 with bandwidths of 25/20 nm on a 2104 EnVisionV Multilabel
Plate Reader which had EnVision Workstation version 1.7
(PerkinElmer, Waltham, MA).
Monoamine oxidase A and B
Cytochrome P450 1A2
The protein in addition to the reagents for the chromogenic solution
(vanillic acid (4-hydroxy-3-methoxylbenzoic acid, 97% purity), 4-
aminoantipyrine (reagent grade), horseradish peroxidase, and the
substrate tyramine hydrochloride (minimum 99% purity)) as well as
the potassium phosphate buffering agents (potassium phosphate
dibasic trihydrate (ꢁ99% ReagentPlusTM) and potassium phosphate
monobasic (minimum 98% purity, molecular biology tested)) were
all purchased from Sigma-Aldrich (St. Louis, MO). The protocol of
continuous spectrophotometric assay by Holt et al. was first used to
determine the activity of the proteins23. The assay was performed in
0.2M potassium phosphate buffer pH 7.6 on 94-well plates (NuncTM
96 F microwell plate without a lid, Nunc A/S, Roskilde, DK) with
chromogenic solution containing 250 mM vanillic acid, 125 mM 4-
aminoantipyrine and 2 U/ml horseradish peroxide in the total assay
volume of 200 ml. The protein was first incubated for 30min at 37ꢃC
in the chromogenic solution and then the substrate tyramine was
introduced at 0.5mM final plate concentration completing the assay
volume. The activity measurement using multilabel reader (VictorTM
Inhibition of CYP1A2 activity was determined using commercial
heterologously expressed human CYP1A2 enzyme (Corning Inc.,
Corning, NY) essentially as described previously24.
Molecular docking
The small-molecule ligands (Figure 2), including their probable
tautomeric states and 3D conformers, were built using LIGPREP,
CONFGEN, and MACROMODEL modules in MAESTRO 2016–3
€
(Schrodinger, LLC, New York, NY, 2016) to match pH 7.4. The com-
pounds were docked to the X-ray crystal structures of HSD1 (PDB:
3HB525; Figures 3 and 4), aromatase (PDB: 3EQM26; Figure 6(C)),
MAO-B (PDB: 2V6127; Figure 6(A)) and CYP1A2 (PDB: 2HI428;
Figure 6(B,C)) with the PANTHER protocol29, where the ligand-
binding site is described as a negative image, and the shape and
electrostatic potentials of the Panther-models and ligand confor-
X4, 2030 Multilabel Reader, PerkinElmer, Waltham, MA) at A490 mations are compared using SHAEP30.

748
S. NIINIVEHMAS ET AL.
Figure 3. The canonical binding modes of 3-phenylcoumarin analogues inhibiting 17-b-hydroxysteroid dehydrogenase 1. The H-bonding (magenta dotted lines) and
halogen bonding/favourable electrostatic (green dotted lines) interactions of (A) compounds 2, (B) 5, (C) 7, (D) 6, (E) 4, and (F) 3 shown as suggested by docking. The
active site residues of HSD1 enzyme (stick models with black backbone) bonding with the 3-phenylcoumarin analogues (ball-and-stick models with green backbone)
are shown. The fluorine atom in 6 (D) and 7 (C) as well as the chlorine atom in 4 (E) are shown with pink and orange colour, respectively. Note that the His222 side
chain is set epsilon protonated in order to facilitate H-bonding with the analogues. This is the opposite arrangement, if compared to the delta protonation of His222
suggested by the original E2B-bound HSD1 X-ray crystal structure (PDB: 3HB5)25.
Figure 4. The canonical vs. non-canonical binding mode of compound 1. (A) The “canonical” binding mode at the HSD1 active site, likely adopted by the other 3-phe-
nylcoumarin analogues (Figure 3), is not suggested for compound 1 based on the docking-based SAR analysis; (B) instead, an alternative “non-canonical” pose is pro-
posed for this potent inhibitor. Note that the His222 side chain is set delta protonated to facilitate H-bonding with the analogue’s R3-hydroxyl. See Figure 3 for
further details.
Figure preparation
using organic synthesis38–40. The latter approach was applied here
to demonstrate that 3-phenylcoumarin (Figure 2) is a suitable non-
steroidal scaffold for building potent and selective HSD1 inhibitors.
Figures 2 and 5 showing 2D structures of the 3-phenylcoumarin
scaffold and the analogues are drawn with BIOVIA Draw 2016
ꢀ
(Dassault Systemes, San Diego, CA, 2016). Figures 1, 3, 4 and 6 are
prepared using BODIL31 and VMD 1.9.232
.
Inhibitor design hypothesis
Based on a detailed analysis of the known inhibitors, 3-phenylcou-
marin was chosen as a suitable scaffold for designing non-
steroidal HSD1-specific inhibitors de novo. The analogue ring
system alignment at the active site of HSD1 would mimic the
Results and discussion
Computer-aided drug discovery
Whether the small-molecule design originates from automated vir- hydrophobic packing of the steroid ring (e.g. inhibitor E2B; PDB:
tual screening schemes, expert de novo work33 or combination of 3HB5 (25); Figure 1(D)). The coumarin ring would align in an orien-
the two, the computer-aided drug discovery (CADD) requires experi- tation that allows its C2-carbonyl to form direct hydrogen bonds
mental verification14,34. This is achieved by pairing biochemical (or H-bonds) with the hydroxyl groups of Tyr219 (or Tyr219OH
)
activity testing with, for example X-ray crystallographic studies35, and/or Ser223 (or Ser223OH; Figures 3 and 4). The coumarin ring
site-directed mutagenesis experiments36,37, and/or “mutating” the could flip also sideways, if Arg258 side chain would rotate into the
lead compounds into diverse libraries of closely-related analogues active site to interact with the C2-carbonyl. The probability of this

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
749
Figure 5. The docking-based structure-activity relationship analysis of the 3-phenylcoumarin analogues with 17-b-hydroxysteroid dehydrogenase 1.
Figure 6. The binding of coumarin derivatives with aromatase, monoamine oxidase B and CYP1A2. (A) With the MAO-B (yellow cartoon), the docked pose of 6 demon-
strates the analogous hydrophobic packing characteristic of the 3-phenylcoumarin analogues with the inhibitor C18 (stick model with orange backbone; PDB: 2V61)27.
Notably, the R6-positioned polar group, fluorine in particular, improves the inhibition by forming a halogen bond with the Leu164ꢃ. (B) The docked pose of 4 (ball-and-stick
model with green backbone) at the active site of CYP1A2 (grey cartoon) mimics a-naphthoflavone (stick model with orange backbone; PDB: 2HI4)28. Additionally, the R1-
chlorine packs against the haeme and the C2-carbonyl and R4-hydroxyl, respectively, H-bond with crystal water (wat) and the Thr118OH. (C) Based on docking, 10 (ball-and-
stick model with green backbone) aligns similarly on top of the haeme (CPK model with cyan carbon atoms) in the active site of aromatase (magenta cartoon) as the
androstenedione (stick model with orange backbone). Unlike the 3-phenylcoumarins the compound 10 has an acceptor group or the N3’ in the imidazole ring capable of
H-bonding with the neutral Asp309 (PDB: 3EQM)26 and, thus, 3-imidazolecoumarin is a potent aromatase inhibitor. Alternatively, the N3’ of 10 could be coordinated with
the haeme (not shown). (D) The coumarin ring of 10 is aligned in a way that its C2-carbonyl accepts an H-bond from the Thr124OH. Moreover, the deprotonated and elec-
tronegative N3’ of 3-imidazole ring is likely coordinated with the positively charged iron in the haeme (CPK model with cyan carbon atoms). See Figure 3 for further details.
rotamer adjustment is difficult to estimate due to missing density Inhibition of 17-b-hydroxysteroid dehydrogenase 1 by the
data on the relevant X-ray crystal structure (PDB: 1EQU)41. Beyond 3-phenylcoumarin analogues
this hypothesis, the plan was to establish and improve the 3-
The activity measurements (Table 1) indicated that the 3-
phenylcoumarin binding and HSD1 inhibition by introducing a
phenylcoumarin is indeed
a suitable scaffold for building
number of polar (hydroxyl/methoxy/halogen) moieties for the 3-
phenyl ring’s R4–R6 and the coumarin ring’s R1–R3 positions
(Figure 2; Table 1).
HSD1 inhibitors. The dissimilarities in the inhibition levels between
the analogues arise from their R1–R6 substituents (Figure 1).

750
S. NIINIVEHMAS ET AL.
Five analogues produced ꢁ62% HSD1 inhibition at 5 mM (Table 1). cannot coordinate as many or as strong interactions in this pos-
ition as a hydroxyl (Figure 3(A,C)). However, the addition of R6-
fluorine next to the hydroxyl offsets in part the negative effect of
the R1 substitution. The fluorine is able to form a halogen bond
with His222 (Figure 3(C)). In addition, the R5-hydroxyl of 7 func-
tion in the same dual H-bonding role with the side chains of
Glu283 and His222 (compare to 2 and 5; Figure 3(A–C)).
Moreover, analogues 1, 2 and 4 produced ꢁ68% inhibition (esti-
mated pIC50 ꢁ 6.2) at 1 mM. The most potent inhibitor 4 produced
47% inhibition even at 100 nM. Rest of the analogues elicited
much weaker inhibition at 100 nM. The inhibition was consistently,
regardless of the concentration, more modest for analogues 3, 5,
6 and 7 than for the three most potent analogues. In contrast,
analogues 8 and 9 did not block the HSD1 (Table 1).
When the R1-methoxy of 7 is shifted to the R2 position in 6
(Figure 2), the inhibition is moderately reduced (Table 1). This
effect is analogous to the weakening of inhibition seen in
response to the R1/R2-hydroxyl switch between 2 and 5 (Figure
1(A,B)). The R5-hydroxyl of 6 H-bonds with both His222 and
Glu283 and the adjacent R6-fluorine halogen bonds with His222
(Figure 3(D)). Despite the proximity of the R2-methoxy to several
H-bond donors, such as the Tyr156OH and the Ser143OH, the group
cannot form as coordinated polar interactions as the R1-methoxy
of 7 (Figure 3(C,D)).
The importance of the R1 position is highlighted with 4 (Figure 2)
– the most potent HSD1 inhibitor of the analogues set (Table 1).
Although the R4-hydroxyl of 4 donates an H-bond only to the
Glu283COO- (Figure 3(E)), the inhibition is strong (Table 1). This is
likely due to the hydroxyl/chlorine substitution at the R1 position
(Figure 3(A,E); Table 1) allowing the R1-chlorine to halogen bond
with the Ser143OH and potentially with the Tyr156OH (Figure 3(E)).
Besides, the protons of the Val144N and the Gly145N cater to the hal-
ogen’s negative charge. The inability of 4 to form H-bonds with both
Glu283 and His222 is, therefore, likely offset by the analogue’s ability
to halogen bond (Table 1).
The relatively poor potency of 3 (Figure 2; Table 1) correlates
with its limited ability to H-bond (Figure 2). Although 3 is almost
identical to the most potent inhibitor 4, it lacks the R1-chlorine
(Figure 3(E,F)). The Glu283COO- and the Ser223OH form H-bonds with
the R6-hydroxyl and the C2-carbonyl, respectively (Figure 3(F)). The
Tyr219OH, in turn, is potentially H-bonding with the C2-carbonyl.
This underlines the importance of proximal groups capable of
bonding at the coumarin’s R4-R6 positions for the HSD1 inhibition
(Figure 2; Table 1).
Scaffold hopping: 3-phenylcoumarin vs. steroid alignment
Due to the plasticity of the catalytic site, full understanding of the
structural basis of the HSD1 inhibition or the selectivity is challenging.
The ring systems of the 3-phenylcoumarin could mimic the steroid
ring positioning in four different ways, if only the hydrophobic pack-
ing is considered. To address this issue, a specifically tailored docking
protocol was utilised29,42,43 for predicting how the analogues bind
and elicit the inhibition (Figure 3). This docking-based SAR analysis
point out how the R1–R6 moieties (Figure 2) affect the HSD1 binding
(Table 1) and inhibition (Figures 3 and 4).
Coumarin (2H-chromen-2-one) contains a bicyclic structure of
phenyl ring fused to a six-member ring with 1- and 2-positioned
oxygen atom and carbonyl group, respectively (Figure 2). The 3-
phenyl is tilted in relation to the coumarin ring as indicated by the
small-molecule X-ray crystallography (CSD: QECNUJ)44. The binding
of the 3-phenylcoumarin analogues is predicted to mimic closely
the pose and hydrophobic packing of the E2B’s steroid ring at the
active site of HSD1 (Figure 1(D)). The ring positioning is likely highly
similar or “canonical” for the HSD1 analogues (Figure 3), except for
1 (Figure 4). Moreover, both the Ser223OH and the Tyr219OH are
predicted to H-bond with the coumarin’s C2-carbonyl (Figures 3
and 4).
R1 position is important for strong 3-phenylcoumarin inhibition
A docking-based SAR analysis (Figure 5) explains the atomistic
determinants of the HSD1 inhibition for each analogue.
The strong potency of 2 (Figure 2; Table 1) reflects its ability to
form well-coordinated H-bonds between the proximal R1/R5-
hydroxyl groups and the residues lining both ends of the binding
site (Figure 3(A)). The R1-hydroxyl of the coumarin ring H-bonds
with the main chain oxygen of Tyr156 (or Tyr156O) and the Ser143
side chain. Furthermore, the main chain nitrogen of Val144 (or
Val144N), Gly145N, and Cys186O are favourably positioned in rela-
tion to the analogue’s R1-hydroxyl group. In turn, the R5-hydroxyl
H-bonds with the carboxyl group of Glu283 (or Glu283COO-) and,
reciprocally, accept an H-bond from the epsilon position of His222.
When the R1-hydroxyl of 2 is switched to the R2 position at
the coumarin ring in 5 (Figure 2), the HSD1 inhibition lowers dra-
matically (Table 1). This highlights the importance of the R1 pos-
ition for the 3-phenylcoumarin binding as the overall alignment of
2 and 5 is likely similar despite the switch (Figure 3(A,B)).
Although the R2-hydroxyl is able to H-bond with the Tyr156OH, it
is evident that the R1-hydroxyl of 2 form stronger interactions
with the close-by residues than the R2-hydroxyl (Figure 3(A,B)).
The R5-hydroxyl of 5 assumes the same dual H-bonding role with
Glu283 and His222 as the equivalent hydroxyl of 2; assuring inhib-
ition despite the R1/R2-hydroxyl switch (Table 1).
R5/R6-hydroxyl group is critical for the
3-phenylcoumarin inhibition
Analogues 2–7 (Figure 3; Table 1) collectively indicate that a halo-
gen or hydroxyl at the R1 position (Figure 2) improves the HSD1
inhibition of the 3-phenylcoumarins (Table 1; Figure 5). The abso-
lute position or even the presence of this group is, however, not
essential for inhibition (Figure 2; Table 1). In contrast, if one con-
siders only those six analogues (Figure 3), excluding 1 (Figure 4),
that produces inhibition at 1 or 5 mM (Table 1), placing a hydroxyl
group at the R4 or R5 position is a necessity (Figure 2).
In 8 (Figure 2), there is a hydroxyl group at the R2 position of
the coumarin ring the same way as in 5, but the lack of a hydroxyl
group in the 3-phenyl ring renders the analogue unable to bond
with Glu283 and His222. The loss of this dual contact is not fully
compensated by the R5-fluorine and, as a result, the HSD1 inhib-
ition is non-existent (Table 1). Further evidence of the importance
of R4/R5-hydroxyl is provided by the inability of 9 (Figure 2) to
prevent the HSD1 activation. The R5-methoxy of 9 cannot estab-
lish as strong H-bonding coordination for the 3-phenyl as a
hydroxyl group in the “canonical” pose would. In this respect, 1
(Figure 2) is a noteworthy exception. Although the analogue’s 3-
phenyl ring contains only R5-methoxy group and no hydroxyl moi-
Replacing the R1-hydroxyl with a methoxy lowers the HSD1
inhibition considerably (Figure 5). This effect is apparent when 7
(Figure 2) is compared to 2 (Table 1). Although the R1-methoxy is
H-bonding with the Ser143OH in the docked pose (Figure 3(C)), it ety (Figure 2), it still induces strong inhibition (Table 1).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
751
R3-hydroxyl reverses the 3-phenylcoumarin binding
two (or of all the tested analogues), the close to optimal H-
bonding coordination with the R1- and R5-hydroxyls of 2 inside
the HSD1 active site (Figure 3(A)) could be the underlying reason
for its higher selectivity. However, the lack of 3D structural data
on HSD2 or its homologous proteins, especially regarding the
enzyme’s binding site, make it difficult to resolve this issue.
The binding of 1 is predicted to differ markedly (Figure 4) from
other 3-phenylcoumarin analogues (Figure 3) producing HSD1
inhibition (Table 1; Figure 5). Instead of the “canonical” pose
(Figure 4(A)), the coumarin and 3-phenyl ring systems of 1 are
suggested to have reverse order or “non-canonical” positioning of
at the site (Figure 4(B)) when compared to the other analogues
(Figure 3). Even though this flip represents a profound change for
the scaffold, it imposes only few drawbacks.
The ring systems of 1 pack against the same residues as they
would in the “canonical” pose. Importantly, the R3-hydroxyl
accepts an H-bond from the delta position of His222 (Figure 4(B)).
This interaction is not feasible, when the hydroxyl is switched to
the R2 position to produce the otherwise identical (but inactive)
analogue 9 (Figure 2). Moreover, the C2-carbonyl and the hetero-
cyclic oxygen in the coumarin can H-bond with the Tyr219OH in
this “non-canonical” pose. Due to the flip, the Glu283COO- cannot
H-bond with 1 (Figure 4); however, the inward pose of the residue
is not required for binding (PDB: 3KLM45). In this “non-canonical”
pose, the R5-methoxy H-bonds with the Ser143OH and, addition-
ally, the Val144N and Tyr156OH are favourably oriented towards
the polar group (Figure 4(B)).
Monoamine oxidases (MAO) A and B are inhibited to some
degree by the 3-phenylcoumarin analogues and this effect is not-
able for the MAO-B (see e.g.18,48,49). For that reason, the inhibition
levels of the analogues were studied here against both enzyme
subtypes (Table 1). Analogous to earlier studies18,49–51 analogues
showing HSD1 inhibition also blocked the MAO-B activity at
10 mM. However, of the HSD1 inhibitor analogues presented in this
study, only 6 (Figure 6(A)) and 7 have pIC50 above 6 (IC50 < 1 mM).
Based on the docking, the R6-fluorine and R5-hydroxyl of 6 form a
halogen bond and an H-bond with the Leu164O and the Pro102O,
respectively. Interestingly, 4 has been tested with MAO-A and
MAO-B earlier (C6 in18). Although 4 reached 64% inhibition at
10 mM in our studies, it has shown more promising activity in a
study by Delogu et al.18. Overall, the results suggest that the
MAO-B inhibition would not be a critical issue for the new ana-
logues or at least for the most potent of them.
Cytochrome P450 (CYP) enzymes metabolise majority of oestro-
gens first in the liver. In this vital process, CYP1A2 enzyme has a
prominent role52 and, therefore, its unintended inhibition by a
small-molecule could promote upswing in the effective E2 levels.
Because the ultimate goal of any HSD1 inhibitor, including the 3-
phenylcoumarins presented in this study, is to lower the E2 levels
in vivo, their ability to block the CYP1A2 was studied as well. All of
the analogues block CYP1A2 activity at some concentration, how-
ever, only the most potent HSD1 inhibitor 4 blocks its function at
an alarming level (Table 1). The ligands that bind into the narrow
and hydrophobic active site of CYP1A2 can be either substrates
that are metabolised by the enzyme or inhibitors that block its
function. As the substrates can be metabolised at different posi-
tions, it is unpractical to offer just one binding pose for each ana-
logue. Regardless, for example the binding pose of 4, which is the
strongest CYP1A2 inhibitor of the analogue set (Table 1), likely
reminds the validated pose of a-naphthoflavone (Figure 6(B))28.
Based on the docking, the R1-chlorine of 4 packs against the
haeme and the 3-phenyl ring is sandwiched between the side
chains of Phe226 and Phe260 (not shown). Moreover, the C2-car-
bonyl of 4 forms an H-bond with a crystal water the same way as
is seen for a-naphthoflavone and the Thr118OH accepts an H-bond
from the R4-hydroxyl (Figure 6(B)).
Aromatase (CYP19A1) inhibitors are used in breast cancer treat-
ments, but unlike in the case of ER, their potential ability to bind
into both HSD1 and aromatase could not be harmful. Aromatase
inhibitors are predominantly used with post-menopausal breast
cancer patients, because the E2 production via the aromatase
pathway happens locally rather than relying on the ovaries53. In
contrast, although the 3-phenylcoumarin scaffold mimics the ster-
oidal core, and fit into the active site of the aromatase, analogues
1–9 do not produce aromatase inhibition (Table 1). A closer
inspection indicates that this lack of activity is due to the inability
of the polar R1–R6 groups to produce favourable interactions at
the aromatase’s active site. On the one hand, the R1-positioned
chlorine (4; Figure 2), methoxy (7; Figure 2), or hydroxyl group (2;
Figure 2) could bond with the proton of Met374N. On the other
hand, while the R4-hydroxyl groups of 2, 3 and 4 are within the
H-bonding range from the Asp309 side chain, this key residue is in
a neutral state at pH 7.4 (PDB: 3EQM)26 and, therefore, ready to
Cross-reactivity of the 3-phenylcoumarin analogues
It is not enough that drug candidates bind into their target pro-
teins to elicit desired effects in situ. One also needs to consider
their absorption, distribution, metabolism, and excretion (ADME)
properties, toxicity, off-target effects, and overall selectivity. For
example coumarins are known to produce hepatotoxic effects
with a certain subgroup of humans – a phenomenon likely emerg-
ing from problems in the 7-hydroxylation of coumarins by the
genetically polymorphic CYP2A6 enzyme46,47. Although no animal
testing was performed in this study, the cross-reactivity of the 3-
phenylcoumarin analogues was tested against ER, HSD2, CYP1A2,
MAO-A, MAO-B, and aromatase using in vitro assays (Table 1).
Oestrogen receptor (ER) antagonists/agonists or selective oes-
trogen receptor modulators, such as tamoxifen and raloxifene are
used routinely in treatment against ER-positive breast cancer.
Potent HSD1 inhibitors could have a dual function as ER antago-
nists but they should not have a dual role as ER agonists promot-
ing breast tissue tumourigenesis. Thus, the effect of the HSD1
inhibitor analogues was studied against both ER and of the potent
HSD1 inhibitor analogues, only 5 was found to produce moderate
ER inhibition. Of the more modest HSD1 inhibitor analogues 6
yielded reasonable ER inhibition. In addition, compounds 8 and 9,
which do not inhibit HSD1 activity, inhibited ER. The molecular
basis for this is clear based on a prior study with the ER-specific
compounds14: the 3-phenylcoumarin scaffold must have R2-func-
tional group, e.g. R2-hydroxyl moiety, at its coumarin ring system
to produce the inhibitory effect.
17-b-hydroxysteroid dehydrogenase 2 (HSD2), which is the
enzymatic counterpart of HSD1, converts E2 to E1. Accordingly, to
avoid counterproductive effects, it is paramount that any potential
drugs aiming to lower the E2 production should not effectively
block the HSD2 activity as a side effect. The activity testing indi-
cates that none of the 3-phenylcoumarin analogues produce >50%
HSD2 inhibition at 1 mM as the inhibition remains at a range from
7 to 42% (Table 1). Notably, the most potent HSD1 inhibitor ana-
logues block the HSD2 only at a moderate level (1 at 27%, 2 at
7%, and 4 at 16%; Table 1). If concentrating on the HSD2 activity,
2 is the most selective HSD1 inhibitor analogue while 4 is a close
runner-up. Although 4 is the more potent HSD1 inhibitor of the donate a proton instead of accepting one.

752
S. NIINIVEHMAS ET AL.
3-Imidazolecoumarin inhibits aromatase potently
2, and 4; Figure 2; Table 1). The approximated pIC50 value at 1 mM
for the three best analogues was ꢁ6.2. Housing polar moieties at
the R5 and/or R6 positions in the 3-phenyl ring is generally critical
for establishing the 3-phenylcoumarin binding and inhibition with
HSD1 (Figure 5; Table 1). Introducing yet another polar group at
the R1 position (Figure 5) in the coumarin ring boosts the HSD1
inhibition even further (e.g. 4; Figure 3(E); Table 1). Moreover,
inserting a hydroxyl group at the R3 position is expected to
reverse the 3-phenylcoumarin binding at the active site (Figure 5)
in comparison to the other analogues (1; Figures 3 and 4(B)) but
without doing away with the inhibition (Table 1). A thorough
cross-reactivity analysis highlights the fact that the 3-phenylcou-
marin analogues block HSD2 only at moderate levels (Table 1),
which is an essential feature for any potential drug candidates aim-
ing to combat the E2-linked diseases, such as breast cancer and
endometriosis. In addition, substituting the 3-phenyl with an imid-
azole changed the scaffold selectivity completely as the resulting
compound 10 blocked potently the aromatase instead of the HSD1.
To sum up, the coumarin core can be tailored to block the E2 syn-
thesis by either the sulphatase pathway or the aromatase pathway
by adding either a 3-phenyl or a 3-imidazole ring, respectively.
The analysis of analogues 1–9 (see above) indicated that the couma-
rin-based compounds with flat ring systems at the 3-position could
fit into the active site of the aromatase. However, a simple H-bond
acceptor at the R4 position, such as a carbonyl group (of androstene-
dione in Figure 6(C)) would be needed to avoid the detrimental clash
of proton donors between the bound ligand and the neutral Asp309
side chain at the active site. Instead of trying to “mutate” 3-phenyl-
coumarin core further to facilitate aromatase inhibition, a new kind
of coumarin-derivative 10, in which the 3-phenyl ring is substituted
with a 3-imidazole was synthesised (Figure 2).
There are two potential binding poses at the aromatase’s active
site for 10. First, the deprotonated N30 of the 3-imidazole ring could
accept an H-bond from the neutral Asp309 side chain (Figure 6(C)).
Second, the N30 could coordinate directly with the haeme.
Although the latter option was not put forward by the docking (not
shown), the imidazole group is known to bind strongly with the
haeme groups and induced-fit effects could help to accommodate
it at the site. Nevertheless, the activity testing shows that 10 inhib-
its strongly the aromatase (pIC50 ¼ 7.11; Table 1).
Furthermore, cross-reactivity testing of 10 indicates that the com-
pound is blocking neither HSD1 nor MAO-B but it has a stronger
inhibitory effect with CYP1A2 than with any of the 3-phenylcoumarin
analogues (Table 1). The coumarin ring of 10 is likely to be flipped in
a reverse pose inside the active site of CYP1A2 in comparison to the
3-phenylcoumarin analogues (Figure 6(B,D)). Importantly, in this pose
the deprotonated and electronegative N3’ of imidazole would be
coordinated with the positively charged iron in the middle of the
haeme; meanwhile, the C2-carbonyl of 10 accepts an H-bond from
the Thr124OH (Figure 6(D)).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
Academy of Finland is acknowledged for funding [Project No.
250311] and The Finnish IT Center for Science (CSC) for computa-
tional resources [Project Nos. jyy2516 and jyy2585].
3-Phenylcoumarins are not pan-assay interference compounds
ORCID
The cross-reactivity data demonstrates that coumarin with C3-
substituted phenyl or imidazole ring does not belong to the pan
assay interference compounds (PAINS) category, but that it is a
privileged structure, which can be fine-tuned or tailored to func-
tion selectively with various targets. The PAINS filtering54, per-
formed using CANVAS module of MAESTRO, supported this
conclusion (no compounds filtered out).
Coumarins are a widely studied group of compounds with struc-
tural and pharmacological variability. Thus, it is not surprising that
also 3-phenylcoumarins have been studied against other targets
elsewhere. Some of the compounds published here have been pre-
viously tested for inhibitory activity against for HIV-1 replication (1;
C17 in15), immune complex-mediated neutrophil oxidative metabol-
ism (2; CHEMBL486894; C13 in16), glyceraldehyde-3-phosphate
dehydrogenase (3; CHEMBL71407; C18 in17), and MAO-A and -B (4;
C6 in18). All these compounds showed moderate ability to inhibit
their intended targets. This further shows that 3-phenylcoumarins
have interesting pharmacologic properties and that they have a
broad utilisation range over therapeutic target proteins.
Pekka A. Postila
Sanna Rauhamaki
Olli T. Pentikainen
http://orcid.org/0000-0002-2947-7991
http://orcid.org/0000-0002-3014-3120
http://orcid.org/0000-0001-7188-4016
€
€
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