Synthesis and biochemical evaluation of warhead-decorated psoralens as (Immuno)proteasome inhibitors

Article abstract of DOI:10.3390/molecules26020356

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibit

Full text of DOI:10.3390/molecules26020356

molecules
Article
Synthesis and Biochemical Evaluation of Warhead-Decorated
Psoralens as (Immuno)Proteasome Inhibitors
Eva Shannon Schiffrer 1, Matic Proj 1 , Martina Gobec 1, Luka Rejc 2, Andrej Šterman 1 , Janez Mravljak 1
,
1,
Stanislav Gobec 1 and Izidor Sosicˇ
*
1
Faculty of Pharmacy, University of Ljubljana, Aškercˇeva cesta 7, SI-1000 Ljubljana, Slovenia;
eva.shannon.schiffrer@ffa.uni-lj.si (E.S.S.); matic.proj@ffa.uni-lj.si (M.P.); martina.gobec@ffa.uni-lj.si (M.G.);
andrej.sterman@ffa.uni-lj.si (A.Š.); janez.mravljak@ffa.uni-lj.si (J.M.); stanislav.gobec@ffa.uni-lj.si (S.G.)
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecˇna pot 113,
1000 Ljubljana, Slovenia; luka.rejc@fkkt.uni-lj.si
2
*
Correspondence: izidor.sosic@ffa.uni-lj.si; Tel.: +386-1-4769-569
Abstract: The immunoproteasome is a multicatalytic protease that is predominantly expressed in
cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases,
various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities
is therefore a viable approach for the treatment of these diseases. However, the development of
immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task.
We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathia-
zolone warhead as selective inhibitors of the chymotrypsin-like (β5i) subunit of immunoproteasome.
Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen
core on the inhibitory potencies. Despite mapping the chemical space with different warheads,
all compounds showed decreased inhibition of the β5i subunit of immunoproteasome in comparison
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial
information about structure–activity relationships that will serve as guidance for the further design
of (immuno)proteasome inhibitors.
Citation: Schiffrer, E.S.; Proj, M.;
Gobec, M.; Rejc, L.; Šterman, A.;
Mravljak, J.; Gobec, S.; Sosicˇ, I.
Synthesis and Biochemical Evaluation
of Warhead-Decorated Psoralens as
(Immuno)Proteasome Inhibitors.
Molecules 2021, 26, 356. https://
doi.org/10.3390/molecules26020356
Keywords: immunoproteasome; psoralen core; non-peptidic; electrophilic compounds; warhead scan
1. Introduction
In mammals, most intracellular proteins are destined for degradation, which involves
Academic Editor: Maria João Matos
Received: 13 December 2020
Accepted: 9 January 2021
the proteasome, a multiprotease complex [
the ubiquitin-proteasome system that is responsible for the maintenance of protein home-
ostasis and the regulation of various cellular processes [ ]. It is a nucleophilic hydrolase
with N-terminal Thr1 acting as a nucleophile to cleave the peptide bond of proteins [ ].
1–3]. The 26S proteasome represents the heart of
4–6
Published: 12 January 2021
7
The 26S proteasome is comprised of a 20S core particle (CP) and 19S regulatory units.
The 20S core is a 720 kDa large barrel-shaped structure assembled of four stacked rings,
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
each consisting of seven subunits. The two outer
like “gates” allowing the entry of unfolded proteins to the two inner
three catalytically active subunits responsible for proteolysis of substrates [
shows caspase-like activity, subunit 2 trypsin-like activity, whereas subunit
chymotrypsin-like activity [ 10]. There are three individual CP types: the constitutive pro-
α
rings provide structural integrity and act
rings, which contain
]. Subunit
5 exhibits
β
8
β1
β
β
9,
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
teasome (cCP), which is expressed in all eukaryotic cells, the thymoproteasome (tCP) [11],
which is exclusive to cortical thymic epithelial cells, and the immunoproteasome (iCP) [12],
which is expressed in cells of hematopoietic origin, but can also be induced in other tissues.
Namely, the induction of iCP in other cell types is possible during acute immune and
inflammatory responses [13
factor and interferon- causes the expression of the iCP active subunits
β1i, β2i, β5i), which replace their constitutive counterparts [12,16].
–
15]. Exposure to inflammatory factors, such as tumor necrosis
α
γ
β
(designated as
Molecules 2021, 26, 356. https://doi.org/10.3390/molecules26020356
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 356
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Increased expression of cCP and iCP can lead to a number of diseases. These in-
clude many types of cancer, infections, inflammatory and autoimmune diseases (Crohn’s
disease, ulcerative colitis, hepatitis, and rheumatoid arthritis), as well as neurological
disorders [17
–
23]. The cCP and the iCP therefore represent validated targets for the de-
27]. The druggability of both CPs
sign of new pharmacologically active compounds [24
–
is clearly represented by the clinically used covalent inhibitors bortezomib, carfilzomib,
and ixazomib, which are used for the treatment of multiple myeloma and mantle-cell-
lymphoma [27]. Selective inhibition of the iCP’s
β5i [28] subunit or simultaneously acting
on 1i and 5i catalytic activities [29 30] are both approaches that are being investigated
β
β
,
in the treatment of autoimmune and inflammatory diseases. In addition, such strategy
should cause fewer adverse effects, as the expression of iCP is induced during the course
of disease processes [31,32]. By avoiding cCP inhibition, the protein degradation would
thus not be inhibited in most eukaryotic cells.
The most advanced iCP inhibitors that are frequently utilized in functional studies
of iCP inhibition are represented in Figure 1. Please note that only a selected number
of derivatives is depicted; namely, the most studied
1i and
5i dual inhibitors KZR-616 [29] and ‘compound 220 [33], as well as the most
selective 5i inhibitor DPLG-3 [34]. Structurally, these compounds all possess a peptidic
β5i-selective inhibitor PR-957 [28],
β
β
β
backbone. Moreover, the former three compounds are all endowed with an electrophilic
warhead, which reacts with the catalytic Thr1 of the proteasome subunits to form a covalent
bond and to confer improved inhibition [24].
Figure 1. Structures of the most studied iCP-selective peptidic inhibitors. For a more thorough overview on subunit-selective
iCP inhibitors, the reader is referred to recent reviews [32,35].
Because peptidic compounds, such as bortezomib and carfilzomib, are prone to
poor metabolic stability and low bioavailability due to the unfavorable physico-chemical
characteristics [36
Despite being significantly less represented, there were some recent reports on non-peptidic
inhibitors of the iCP (mostly inhibiting the 5i subunit) and the representative compounds
are shown in Figure 2 [39 44]. As with peptidic compounds, irreversible inhibitors of
–38], there is a need to develop inhibitors with non-peptidic scaffolds.
β
–
non-peptidic nature can be obtained through structure-guided optimization, whereby an
electrophilic warhead is properly positioned onto the structure of the non-covalently
binding scaffold [45]. An essential prerequisite for this strategy to work is that the position
of the electrophilic moiety allows the formation of the covalent bond between the inhibitor
and the catalytic Thr1.
Recently, we discovered non-peptidic and β5i-selective inhibitors with a central pso-
ralen core [39]. The most potent non-covalent inhibitor obtained during structure-activity
0
relationship (SAR) studies possessed a phenyl substituent at position 4 (see Figure 3 for pso-
ralen atom numbering). This compound was also transformed into two potent irreversible
covalent inhibitors by adding electrophilic warheads at position 3, i.e., succinimidyl ester
and oxathiazolone. Of these two compounds, the oxathiazolone-based inhibitor showed
the most promising inhibitory characteristics (Figure 2, ‘compound 420) as it was a po-
tent and selective iCP inhibitor [39]. It was demonstrated previously that oxathiazolones
inhibit iCP via cyclocarbonylation of the
β-OH and α-NH2 of the active site Thr1 [41].
Nevertheless, this structural fragment is deemed hydrolytically unstable making it less

Molecules 2021, 26, 356
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suitable for further development [41]. This fact prompted us to investigate other possible
warheads that could be attached at the same position of the psoralen core. Previously,
we already determined that acrylamides and nitrile-based warheads led to worse inhibition
of the iCP [39]. However, to further map the warhead chemical space attached onto the
psoralen core, we prepared a new focused set of compounds with different electrophilic
fragments attached at position 3 (Figure 3), and evaluated their influence on the inhibition
of all six catalytic subunits of both CPs. The selection of warheads in this study was
based both on previously well described Thr targeting warheads (e.g., vinyl sulfones,
α’,β’-epoxyketones) [24], as well as on biologically less represented electrophilic moieties.
In addition, to minimize the influence of non-covalently binding portion of the molecule
on overall inhibitory potency, we used the same core compound with a phenyl substituent
at position 40.
Figure 2. A selection of non-peptidic iCP inhibitors. ‘Compound 42’ [39] was the most selective irreversible β5i subunit
inhibitor from the initial series of psoralen-based inhibitors. It represents the parent compound for studies in this manuscript.
Figure 3. Schematic representation of the work described in this study. The numbering system for the psoralen ring is
shown for clarity, as well as general nomenclature for the warhead moieties used.
2. Results and Discussion
2.1. Syntheses of 3-Substituted Psoralens
To prepare 3-allyl-substituted psoralen, ethyl acetoacetate was used as a starting
material (Scheme 1). It was first alkylated using NaH as a base to obtain compound
which was subjected to Pechmann reaction conditions to yield 7-hydroxycoumarin deriva-
tive . After OH group alkylation with 2-bromoacetophenone, the final allyl-substituted
compound was obtained by base-catalyzed condensation of the coumarin derivative
into psoralen ring (Scheme 1). A compound with 3-vinyl-based warhead attached
at position 3 (compound ) was obtained via a similar route. The crucial intermediate
1,
2
4
3
7

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7-hydroxy-4-methyl-4-vinyl-2H-chromen-2-one (
5
) was obtained in high yield by heat-
◦
ing resorcinol derivative and crotonyl chloride at 60 C in acetone. This was followed
by a 2-bromoacetophenone-mediated alkylation and cyclization into psoralen yielding
compounds 6 and 7, respectively.
Scheme 1. Synthesis of compounds with allyl (
4
) and vinyl◦(
7
) warheads attached at position 3 of the psoralen ring.
◦
Reagents and conditions: (a) allyl bromide, NaH (60%), THF, 0 C to rt, overnight; (b) resorcinol, 98% H2SO4, dioxane, 0 C
to rt, overnight; (c) 2-bromoacetophenone, K2CO3, KI, dioxane, 100 ◦C, 24 h; (d) 1 M NaOH, propan-2-ol, 80 ◦C, 40 min;
◦
◦
(e) crotonyl chloride, K2CO3, acetone, 60 C, 24 h; (f) 1 M KOH, EtOH, 85 C, 2 h.
Compounds and were further used as synthons to prepare derivatives with other
4
7
electrophilic moieties at position 3 (Scheme 2). The former was used in a Wacker-type oxi-
dation of the terminal olefin by the combination of Pd(OAc)2 and Dess-Martin periodinane
to prepare the compound with ethyl methyl ketone moiety, i.e., compound
substituted derivative was a starting point for three different warhead-decorated pso-
ralens, namely vinyl sulfone (via NH4I-induced sulfonylation of vinyl at position 3
with DMSO), 3-bromo-4,5-dihydroisoxazole 10 46] (via cycloaddition of the alkene with
8. The vinyl-
7
9
[
1,1,-dibromoformaldoxime), and pinacolate ester 11 (via transition-metal-free synthesis of
alkylboronate from vinyl and bis(pinacolato)diboron) (Scheme 2).
Scheme 2. Synthesis of compounds 8, 9, 10, and 11 with ketone, vinyl sulfone, 3-bromo-4,5-dihydroisoxazole, and
pinacolate ester, respectively, as warheads. Reagents and conditions: (a) Dess–Martin periodinane, Pd(OAc)2, CH3CN, H2O,
50 ◦C, overnight; (b) DMSO, H2O, NH4I, 130 ◦C, 36 h; (◦c) 1,1-dibromoformaldoxime, DMF, NaHCO3,
−
15 ◦C to rt, 5 h;
(d) bis(pinacolato)diboron, CsF, 1,4-dioxane, MeOH, 100 C, 12 h.

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The synthesis of 3-propanal-substituted psoralen 15 was initiated by a coumarin
derivative 12 possessing ethyl propionate moiety at position 3 (Scheme 3). The acidic
hydrolysis yielded propanoic acid 13, which was transformed into aldehyde deriva-
tive 14 by first forming an acid chloride, followed by in situ reduction with hydrogen
gas using Pd/BaSO4 as a catalyst. Interestingly, an attempt to prepare
α-ketoaldehyde
(which is a known Thr-targeting warhead [24]) from compound 15 by Riley oxidation
with SeO2 resulted in the formation of
Figures 4 and 5).
α,β-unsaturated aldehyde derivative 16 (Scheme 3,
Scheme 3. Synthesis of compounds with aldehyde- (15) and α,β-unsaturated aldehyde-based (16) warheads attached at
position 3 of the psoralen ring. Rea◦gents and conditions: (a) 1 M HCl, dioxane, reflux, 2 h; (b) i. SOCl2, DMF, toluene, rt,
◦
◦
17 h; ii. H2, Pd/BaSO4, toluene, 100 C, 2 h; (c) 1 M NaOH, propan-2-ol, 60 C, 15 min; (d) SeO2, dioxane, H2O, MW, 150 C,
1 h. Synthesis of compound 12 was described previously [39].
Figure 4. COSY experiment for 16. Circled cross-peaks indicate coupling between aldehyde proton CHO and the adjacent
0
0
0
C2 -H, and between C2 -H and C3 -H.

Molecules 2021, 26, 356
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0
Figure 5. NOESY experiment for 16. Only cross-peaks that indicate coupling between CH3 protons and C4-H and C3 -H
are shown.
To further confirm the structure of α,β-unsaturated aldehyde 16, two-dimensional
NMR experiments correlation spectroscopy (COSY) and Nuclear Overhauser effect spec-
troscopy (NOESY) were recorded. In the COSY spectrum (Figure 4), a clear correlation
between the aldehyde proton CH H was observed. In addition, the
O and the adjacent C20-
NOESY experiment showed a coupling between the CH3 protons and C4-
(Figure 5).
H H
and C30-
The fact that the most advanced selective iCP inhibitors and also carfilzomib, which
is a marketed cCP and iCP inhibitor, possess an ’, ’-epoxyketone fragment as the
Thr-targeting warhead, encouraged us to prepare two such psoralen-based compounds
α
β
(Scheme 4). Both 20 and 21 were synthesized from the corresponding precursors 17
and 19 by a HATU-mediated amide bond formation (Scheme 4).
, 18,
◦
Scheme 4. Synthesis of epoxyketone-based compounds 20 and 21. Reagents and conditions: (a) i. TFA, CH2Cl2, 0 C,
30 min; ii. HATU, HOBt H2O, DIPEA, DMF (20) or CH2Cl2 (for 21), rt, 24 h. Syntheses of compounds 17, 18, and 19 were
×
based on previously described procedures [39], compound 17; [47], compound 18; [48], compound 19. All spectral data
(1H-NMR, HRMS) corresponded well to the original reports.
To prepare 3-azetidin-2-one-substituted psoralen 24, a previously synthesized com-
pound 22 was used as a crucial intermediate. It was first N-acylated with 3-bromopropanoyl
chloride to yield 3-bromopropanamide 23, and then cyclized into the
using NaOtBu as a base (Scheme 5).
β-lactam ring by

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Scheme 5. Synthesis of alkyl bromide-based psoralen 23 and psoralen 24 with azetidin-2-one as a warhead. Reagents and
◦
◦
conditions: (a) 3-bromopropanoyl chloride, K2CO3, CH2Cl2, 0 C to rt, 3 h; (b) NaOtBu, DMF, 0 C to rt, 24 h. Synthesis of
compound 22 was described previously [39].
2.2. Biochemical Evaluation
The target compounds were evaluated for their inhibitory potencies on both CPs
(Table 1) using subunit selective fluorogenic substrates (for details, see Materials and
Methods Section). The data were calculated as residual activities (RAs) of individual
subunits of CPs in the presence of 1 µM of each compound. This concentration was used
due to poor solubility of all final compounds at higher concentrations, emphasizing the
need for development of inhibitors with improved solubility. The previously described
oxathiazolone derivative ‘compound 420 and carfilzomib were used as positive control
using the same concentration (1 µM) to enable a better comparison between compounds.
Table 1. Inhibitory potencies of compounds against all catalytically active subunits (
of the iCP and against all catalytically active subunits ( 5, 2, 1) of the human cCP. In the assays,
5i and
β5i,
β2i, and
β1i)
β
β
β
the following substrates were used: Suc-LLVY-AMC for
β
β5; Boc-LRR-AMC for
β2i and
β2;
Ac-PAL-AMC for β1i; Ac-nLPnLD-AMC for β1.
β5i
β2i
β1i
β5
β2
β1
Cpd
(RA [%]) 1
(RA [%]) 1
(RA [%]) 1
(RA [%]) 1
(RA [%]) 1
(RA [%]) 1
4
7
8
78 ± 5
76 ± 3
70 ± 0
69 ± 13
62 ± 5
76 ± 12
71 ± 1
65 ± 3
78 ± 0
76 ± 1
74 ± 11
77 ± 7
3 ± 1
100 ± 0
100 ± 0
100 ± 0
109 ± 3
102 ± 2
109 ± 14
103 ± 4
107 ± 3
88 ± 0
95 ± 24
87 ± 15
90 ± 21
76 ± 7
87 ± 19
72 ± 8
94 ± 16
92 ± 21
83 ± 18
81 ± 10
72 ± 1
74 ± 5
1 ± 1
80 ± 21
81 ± 18
78 ± 21
72 ± 18
79 ± 20
66 ± 21
76 ± 20
77 ± 19
76 ± 17
79 ± 14
72 ± 12
63 ± 26
0 ± 0
82 ± 7
86 ± 7
87 ± 7
90 ± 2
86 ± 2
89 ± 4
87 ± 3
83 ± 4
81 ± 6
80 ± 4
89 ± 6
88 ± 5
16 ± 6
99 ± 2
88 ± 0
88 ± 5
89 ± 2
99 ± 5
90 ± 3
87 ± 2
97 ± 2
83 ± 2
79 ± 5
86 ± 4
92 ± 4
89 ± 7
2 ± 2
9
10
11
15
16
20
21
23
24
carf.
‘42’
90 ± 0
113 ± 7
109 ± 7
1 ± 1
5 ± 2
102 ± 5
97 ± 8
52 ± 4
99 ± 8
1
RA values are means from at least three independent determinations. Ac-PAL-AMC, acetyl-Pro-Ala-Leu-7-
amino-4-methylcoumarin; Ac-nLPnLD-AMC, acetyl-Nle-Pro-Nle-Asp-AMC; Boc-LRR-AMC, t-butyloxycarbonyl-
Leu-Arg-Arg-7-amino-4-methylcoumarin; Suc-LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin.
carf.: carfilzomib.
Given the fact that all assayed compounds possessed the same non-covalently binding
portion, we were able to thoroughly assess the contributions of attached warheads to the
inhibition of all catalytically active subunits of iCP and cCP. The assay results showed that
all new psoralens were worse inhibitors of β5i subunit of iCP in comparison to the parent
oxathiazolone-based ‘compound 420 (Table 1, Figure 6). This is most probably due to the
mispositioning of the electrophilic carbons of all compounds and the catalytic Thr1Oγ in
the
at 1
β
5i active site. Interestingly, all compounds inhibited
M with RA values ranging from 62 to 78%. Of the 12 prepared compounds, 3-bromo-
-unsaturated
aldehyde as the warhead were the most promising. The former showed RA value of
β5i activity with a similar potency
µ
4,5-dihydroisoxazole-substituted psoralen 10 and compound 16 with an α,β

Molecules 2021, 26, 356
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62 ± 5%, whereas for the latter RA was determined at 65
±
3% (see also postulated binding
modes for 10 and 16 in Figure 7). It was not surprising to see that all 12 compounds also
exhibited worse inhibition of the 5 subunit of cCP, albeit these differences were much less
pronounced as for the 5i subunit. Of note, compounds 11 23, and 24 were slightly better
inhibitors of
1i subunit of iCP in comparison to the ‘compound 420. All psoralen-based
compounds (with oxathiazolone included) did not inhibit other subunits of both CPs (i.e.,
2i, 2, and 1), whereas carfilzomib completely abolished activity of all subunits at 1
(Table 1, Figure 6).
β
β
9
,
,
β
β
β
β
µM
Figure 6. Inhibition results represented as bar charts of inhibition percentage. carf.: carfilzomib.
Figure 7. Molecular modelling. Binding site residues are presented as green sticks with labels for some of the key residues.
(
A
) Covalent docking of ‘420 (magenta) into the
β5i subunit (PDB: 5M2B). Please note that only the initial intermediate
formed after the nucleophilic attack of OH group of Thr1 onto the carbonyl group of the oxathiazolone is represented.
Co-crystalized ligand Ro19 is presented with blue sticks and dashed yellow lines for hydrogen bonds. (
docking of 10 (cyan) and 16 (yellow) reveals good alignment of the psoralen core with the proposed pose of ‘42 (magenta).
However, the distance from the electrophilic carbons of 10 and 16 to the catalytic Thr1Oγ is too large to form a covalent bond.
B
) Noncovalent
0

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3. Materials and Methods
3.1. General Chemistry Methods
Reagents and solvents were obtained from commercial sources (Acros Organics
(Thermo Fisher Scientific, Waltham, MA, USA), Sigma-Aldrich (Merck KGaA, Darmstadt,
Germany), TCI Europe (Tokyo Chemical Industry, Tokyo, Japan), Alfa Aesar (Thermo Fisher
Scientific, Waltham, MA, USA), Fluorochem (Fluorochem Ltd., Derbyshire, UK) and were
used as received. Carfilzomib were purchased from MedChemExpress. For reactions
involving air or moisture sensitive reagents, solvents were distilled before use and these re-
actions were carried out under nitrogen or argon atmosphere. Reactions using microwaves
were performed on a standard monomode microwave reactor MONOWAVE 200 (Anton
Paar, Graz, Austria). Reactions were monitored using analytical thin-layer chromatog-
raphy plates (Merck 60 F254, 0.20 mm), and the components were visualized under UV
light and/or through staining with the relevant reagent. Normal phase flash column
chromatography was performed on Merck Silica Gel 60 (particle size 0.040–0.063 mm;
1
Merck, Germany). H and 13C-NMR spectra were recorded at 295 K on a Bruker Avance III
400 MHz spectrometer (Bruker, Billerica, MA, USA) operating at frequencies for 1H-NMR
at 400 MHz and for 13C-NMR at 101 MHz. The chemical shifts (
δ) are reported in parts per
million (ppm) and are referenced to the deuterated solvent used. The coupling constants
(J) are given in Hz, and the splitting patterns are designated as follows: s, singlet; br s,
broad singlet; d, doublet; app d, apparent doublet; dd, doublet of doublets; ddd, doublet of
doublets of doublets; t, triplet; dt, doublet of triplets; td, triplet of doublets; m, multiplet.
All 13C-NMR spectra were proton decoupled. Mass spectra data and high-resolution mass
measurements were performed on a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-
Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The purity of
the compounds used in biochemical assays was determined with analytical normal-phase
HPLC on an Agilent 1100 LC modular system (Agilent, Santa Clara, CA, USA) that was
equipped with a photodiode array detector set to 254 nm. A Kromasil 3-CelluCoat column
(150 mm
a sample injection volume of 5–20
(isopropanol) was used; the ratio used is described for each compound below. The puri-
×
4.6 mm; 3
µ
m particle size) was used, with a flow rate of 1.0 mL/min and
µL. An isocratic eluent system of A (hexane) and B
ties of the test compounds used for the biological evaluations were
otherwise.
≥
95%, unless stated
3.2. Syntheses
Synthesis of ethyl 2-acetylpent-4-enoate (1):
To a solution of ethyl acetoacetate (7.28 mL, 7.50 g, 57.60 mmol, 1 equiv.) in 50 mL
of anhydrous THF, NaH in mineral oil (60%, 2.30 g, 57.60 mmol, 1 equiv.) was added
and the resulting suspension stirred under argon at 0 ◦C. After 20 min, a solution of allyl
bromide (4.99 mL, 6.97 g, 57.60 mmol, 1 equiv.) in 25 mL of anhydrous THF was added
dropwise. The reaction mixture was stirred at room temperature overnight. Next, cold H2O
(25 mL) was added and THF was evaporated under reduced pressure. The resulting
suspension was extracted with Et2O (3
anhydrous Na2SO4, and evaporated. The product was purified by column chromatography
(Et2O/petroleum ether, 1/5, v/v). Yield: 71%, clear liquid. H-NMR (400 MHz, DMSO-d6)
×
25 mL), the organic layer separated, dried over
1
δ
1.17 (t, J = 7.1 Hz, 3H, C
H
3CH2), 2.18 (s, 3H, COC
H
3), 2.42–2.47 (m, 2H, CH2CHC
H2CH),
3.73 (dd, J = 7.8, 6.8 Hz, 1H, C
H
), 4.07–4.15 (qd, 2H, J = 7.1, 1.6 Hz, CH3C
H
2), 4.98–5.10 (m,
2H, CH2CHCH2CH), 5.67–5.77 (m, 1H, CH2CHCH2CH).
Synthesis of 3-allyl-7-hydroxy-4-methyl-2H-chromen-2-one (2):
This compound was prepared using Pechmann condensation as follows. A solution
of resorcinol (4.06 g, 36.90 mmol, 1 equiv.) and ethyl 2-acet◦ylpent-4-enoate (
1) (6.90 g,
40.50 mmol, 1.1 equiv.) in dioxane (80 mL) was cooled to 0 C, followed by drop-wise
addition of concentrated H2SO4 (98%, 19.60 mL, 405 mmol, 10 equiv.). The reaction mixture
was stirred at room temperature overnight. Dioxane was then evaporated under reduced
pressure and the semi-solid mixture was added portion-wise to an ice-cold solution of KOH

Molecules 2021, 26, 356
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(40 g) in H2O (100 mL). The pH was adjusted to 13 with KOH and the resulting white solid
was filtered off. The filtrate was extracted with EtOAc (3
ganic extracts were dried over anhydrous Na2SO4 and evaporated under reduced pressure.
×
25 mL) and the combined or-
The compound was purified by column chromatography (EtOAc/n-hexane, 1/1.5, v/v,
1
dry loading) yielding a pale-yellow solid. Yield: 13%. H-NMR (400 MHz, DMSO-d6)
δ
2.34
(s, 3H, CH3), 3.30 (d, J = 6.0 Hz, 2H, Ar-CH2CHCH2), 4.98–5.06 (m, 2H, Ar-CH2CHCH2),
5.84 (ddt, J = 16.3, 10.3, 6.0 Hz, 1H, Ar-CH2C CH2), 6.70 (d, J = 2.4 Hz, 1H, Ar- ), 6.80 (dd,
), 10.44 (s, 1H, O ); HRMS (ESI) m/z
H
H
J = 8.7, 2.4 Hz, 1H, Ar-
H
), 7.63 (d, J = 8.7 Hz, 1H, Ar-
H
H
calculated for C13H11O3 [M − H]− 215.0714, found 215.0707.
Synthesis of 3-allyl-4-methyl-7-(2-oxo-2-phenylethoxy)-2H-chromen-2-one (3):
This compound was synthesized following a previously described procedure [39].
Briefly, to a solution of 3-allyl-7-hydroxy-4-methyl-2H-chromen-2-one (2) (0.99 g, 4.56 mmol,
1 equiv.) in dioxane (70 mL), K2CO3 (2.52 g, 18.22 mmol, 4 equiv.) and KI (76 mg, 0.46 mmol,
0.1 equiv.) were added. After 10 min of stirring at 100 ◦C, 2-bromoacetophenone (◦1.36 g,
6.83 mmol, 1.5 equiv.) was added and the mixture was further stirred at 100 C for
24 h. The solvent was then removed under reduced pressure, followed by addition of
H2O (30 mL) to the residue. The aqueous phase was extracted with EtOAc (3
the combined organic extracts were washed with brine (20 mL), dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure. The compound was purified by
×
30 mL),
1
crystallization from MeOH yielding pale-yellow crystalline solid. Yield: 67%. H-NMR
(400 MHz, DMSO-d6)
J = 8.5, 3.0, 1.3 Hz, 2H, Ar-CH2CHC
1H, Ar-CH2C CH2), 7.03 (dd, J = 8.9, 2.6 Hz, 1H, Ar-
7.59 (app dd, J = 10.6, 4.8 Hz, 2H, 2 Ar- ), 7.68–7.77 (m, 2H, 2
); HRMS (ESI) m/z calculated for C21H19O4 [M + H]+ 335.1280,
δ
2.38 (s, 3H, C
H
H
3), 3.32 (d, J = 6.0 Hz, 2H, Ar-C
2), 5.75 (s, 2H, C 2), 5.86 (ddt, J = 16.2, 10.2, 6.0 Hz,
), 7.07 (d, J = 2.5 Hz, 1H, Ar- ),
Ar- ), 8.04 (app dd,
H2CHCH2), 5.03 (ddd,
H
H
H
H
×
H
×
H
J = 8.4, 1.2 Hz, 2H, 2
found 335.1272.
×
Ar-H
Synthesis of 6-allyl-5-methyl-3-phenyl-7H-furo[3,2-g]chromen-7-one (4):
This compound was◦synthesized following a previously described procedure [39].
Namely, to a heated (80 C) and stirred solution of
3 (0.25 g, 0.75 mmol, 1 equiv.) in
propan-2-ol (25 mL), an aqueous solution of NaOH (7.5 mL, 1 M, 10 equiv.) was added.
◦
The reaction mixture was stirred at 80 C for 40 min. After the reaction was complete
(monitored by TLC), propan-2-ol was evaporated under reduced pressure. The aqueous
residue was acidified with HCl (6 mL, 1 M) to pH 5, then H2O (20 mL) was added,
the aqueous layer was extracted with CH2Cl2 (3
×
25 mL), and the combined organic
extracts were evaporated under reduced pressure. The compound was purified by column
chromatography (Et2O/petroleum ether, 1/3, v/v). White solid, yield: 70%. 1H-NMR
(400 MHz, DMSO-d6)
(m, 2H, Ar-CH2CHCH2), 5.89 (ddt, J = 16.1, 10.2, 6.0 Hz, 1H, Ar-CH2C
(m, 1H, Ar- ), 7.51–7.60 (m, 2H, 2 Ar- ), 7.79 (s, 1H, Ar- ), 7.80–7.82 (m, 1H, Ar-
7.83 (t, J = 1.6 Hz, 1H, Ar- ), 8.18 (s, 1H, Ar- ), 8.48 (s, 1H, Ar-
); 1H-NMR (400 MHz,
CDCl3) 2.49 (s, 3H, CH3), 3.49 (d, J = 6.0 Hz, 2H, Ar-CH2CHCH2), 5.01–5.16 (m, 2H,
Ar-CH2CHCH2), 5.94 (ddt, J = 16.2, 10.1, 6.0 Hz, 1H, Ar-CH2C CH2), 7.44 (ddd, J = 7.4,
), 7.51–7.57 (m, 2H, 2 Ar- ), 7.61–7.64 (m,
), 8.00 (s, 1H, Ar-
); 13C-NMR
δ
2.54 (s, 3H, C
H
3), 3.40 (d, J = 6.0 Hz, 2H, Ar-C
H
2CHCH2), 5.01–5.11
CH2), 7.41–7.48
),
H
H
×
H
H
H
H
H
H
δ
H
4.0, 1.3 Hz, 1H, Ar-
1H, Ar- ), 7.65 (t, J = 1.7 Hz, 1H, Ar-
(101 MHz, DMSO-d6)
H
), 7.49–7.51 (m, 1H, Ar-
H
×
H
H
H
), 7.82 (s, 1H, Ar-
H
H
δ
15.19, 30.94, 99.48, 115.69, 116.46, 116.99, 121.18, 121.29, 122.81,
127.23, 127.84, 129.22, 130.72, 134.50, 144.29, 148.39, 149.94, 155.92, 160.58; HRMS (ESI) m/z
calculated for C21H17O3 [M + H]+ 317.1172, found 317.1166. Purity by HPLC (0–18 min;
70% n-hexane/isopropanol): 99%.
Synthesis of 7-hydroxy-4-methyl-4-vinyl-2H-chromen-2-one (5):
A suspension of 1-(2,4-dihydroxyphenyl)ethan-1-one (502 mg, 3.3 mmol, 1 equiv.),
crotonyl chloride (395
µ
L, 429 mg, 4.1 mmol,◦1.25 equiv.) and K2CO3 (1.47 g, 10.6 mmol,
3.2 equiv.) in acetone (25 mL) was heated at 60 C for 24 h. The solvent was then evaporated
under reduced pressure, followed by the addition of EtOAc (100 mL). The organic phase
was extracted with H2O (100 mL), and the aqueous phase acidified with 2 M HCl and

Molecules 2021, 26, 356
11 of 18
further extracted with EtOAc (2
over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The compound
×
100 mL). The combined organic extracts were dried
was purified by column chromatography (EtOAc/n-hexane, 1/4, v/v). White solid, yield:
71%. 1H-NMR (400 MHz, DMSO-d6)
δ
2.45 (s, 3H, CH3), 5.51 (dd, J = 12.0 Hz, 2.4 Hz,
1H, Ar-CHCH2), 6.02 (dd, J = 17.4 Hz, 2.4 Hz, 1H, Ar-CHCH2), 6.67 (d, J = 2.4 Hz, 1H,
Ar- ), 6.72 (dd, J = 17.4, 12.0 Hz, 1H, Ar-C CH2), 6.79 (d, J = 8.9, 2.4 Hz, 1H, Ar- ), 7.66
). HRMS (ESI) m/z calculated for C12H9O3
H
H
H
(d, J = 8.9 Hz, 1H, Ar-H), 10.51 (br s, 1H, OH
[M − H]− 201.0557, found 201.0549.
Synthesis of 4-methyl-7-(2-oxo-2-phenylethoxy)-3-vinyl-2H-chromen-2-one (6):
This compound was synthesized following a previously described procedure [39]; us-
ing the procedure as for
ing off-white crystalline solid. Yield: 79%. H-NMR (400 MHz, CDCl3)
5
. The compound was purified by crystallization from EtOH yield-
1
δ 2.48 (s, 3H, CH3),
5.38 (s, 2H, OCH2), 5.63 (dd, J = 11.8, 1.9 Hz, 1H, Ar-CH=CH2), 6.04 (dd, J = 17.6, 1.9 Hz,
1H, Ar-CH=CH2), 6.71 (dd, J = 17.6, 11.8 Hz, 1H, Ar-C =CH2), 6.77 (d, J = 2.6 Hz, 1H,
Ar-H), 6.96 (dd, J = 9.0, 2.6 Hz, 1H, Ar-H), 7.49–7.57 (m, 2H, Ar-H), 7.59 (d, J = 8.9 Hz, 1H,
Ar- ), 7.70–7.63 (m, 1H, Ar- ), 7.95–8.04 (m, 2H, 2 Ar-
); 13C-NMR (101 MHz, CDCl3)
15.37, 70.55, 101.43, 112.79, 114.88, 120.10, 122.26, 126.34, 127.99, 128.99, 129.03, 134.14,
H
H
H
×
H
δ
134.26, 146.69, 153.55, 160.18, 160.39, 193.18; HRMS (ESI) m/z calculated for C20H17O4
[M + H]+ 321.1121, found 321.1123.
Synthesis of 5-methyl-3-phenyl-6-vinyl-7H-furo[3,2-g]chromen-7-one (7):
To a solution of 4-methyl-7-(2-oxo-2-phenylethoxy)-3-vinyl-2H-chromen-2-one (6)
(132 mg, 0.4 mmol, 1 equiv.) in EtOH (5 mL), KOH (1.2 mL, 1 M, 1.2 mmol, 3 equiv.) was
added and the reaction mixture stirred at 85 ◦C for 2 h. The solvent was then evaporated,
followed by the addition of H2O (20 mL). The suspension was acidified with concentrated
HCl to pH = 1 and extracted with CH2Cl2 (2
were dried over Na2SO4, filtered, and evaporated under reduced pressure. The product
×
50 mL). The combined organic extracts
was purified by column chromatography (EtOAc/n-hexane, 1/1, v/v). Yellow solid; yield:
78 %; 1H-NMR (400 MHz, CDCl3)
δ
2.60 (s, 3H, CH3), 5.69 (dd, J = 11.8 Hz, 1.8 Hz, 1H,
Ar-CH=CH2), 6.05 (dd, J = 17.7 Hz, 1.8 Hz, 1H, Ar-CH=CH2), 6.77 (dd, J = 17.7, 11.8, 1H,
Ar-C =CH2), 7.42–7.47 (m, 1H, Ar- ), 7.49 (s, 1H, Ar- ), 7.51–7.56 (m, 2H, Ar- ), 7.62–7.66
(m, 2H, Ar- ), 7.82 (s, 1H, Ar- ), 8.04 (s, 1H, Ar- 15.92,
); 13C-NMR (101 MHz, CDCl3)
H
H
H
H
H
H
H
δ
99.74, 116.23, 117.38, 121.23, 122.31, 122.82, 123.98, 127.59, 128.04, 129.19, 129.26, 131.11,
142.79, 146.88, 150.39, 156.67, 160.21; HRMS (ESI) m/z calculated for C20H15O3 [M + H]+
303.1016, found 303.1019. Purity by HPLC (0–18 min; 70% n-hexane/isopropanol): 98%.
Synthesis of 5-methyl-6-(2-oxopropyl)-3-phenyl-7H-furo[3,2-g]chromen-7-one (8):
This compound was synthesized following a previously described procedure [49].
Briefly, to a stirred solution of olefin
4 (158 mg, 0.5 mmol, 1 equiv.) in CH3CN (3.5 mL)
and H2O (0.5 mL), Pd(OAc)2 (5.6 mg, 0.025 mmol, 5 mol %) and Dess-Martin periodi-
nane (254 mg, 0.6 mmol, 1.2 equiv.) were added. The reaction mixture was warmed to
50 ◦C and stirred under an argon atmosphere overnight. The reaction mixture was then
filtered through a small pad of Celite and washed with EtOAc, and the filtrate was concen-
trated. The residue was purified by column chromatography (EtOAc/n-hexane = 1/2, v/v
dry loading). White solid, yield: 40%. 1H-NMR (400 MHz, CDCl3)
2.32 (s, 3H, CH2COC 3),
2.44 (s, 3H, CH3), 3.88 (s, 2H, CH2COCH3), 7.44 (t, J = 7.4 Hz, 1H, Ar- ), 7.48–7.57 (m,
3H, 3 Ar- ), 7.63 (app dd, J = 8.0, 1.0 Hz, 2H, 2 Ar- ), 7.83 (s, 1H, Ar- ), 8.01 (s,
1H, Ar- 16.19, 30.15, 42.42, 100.13, 116.08, 117.27, 118.41,
); 13C-NMR (101 MHz, CDCl3)
,
δ
H
H
×
H
×
H
H
H
δ
122.51, 124.19, 127.77, 128.19, 129.41, 131.26, 143.01, 149.65, 150.81, 156.85, 161.93, 204.58;
HRMS (ESI) m/z calculated for C21H17O4 [M + H]+ 333.1121, found 333.1127. Purity by
HPLC (0–18 min; 70% n-hexane/isopropanol): 98%.
Synthesis of (E)-5-methyl-6-(2-(methylsulfonyl)vinyl)-3-phenyl-7H-furo[3,2-g]chromen-
7-one (9):
To a solution of 5-methyl-3-phenyl-6-vinyl-7H-furo[3,2-g]chromen-7-one (7) (100 mg,
0.33 mmol, 1 equiv.) in DMSO (1 mL), H2O (0.5 mL) and NH4I (191 mg, 1.32 mmol,
4 equiv.) were added. The reaction mixture was stirred at 130 ◦C for 36 h. Then, it was

Molecules 2021, 26, 356
12 of 18
cooled to room temperature, followed by slow addition of Na2S2O3
×
5H2O until the
discoloration of mixture. Subsequently, H2O (20 mL) was added and the aqueous phase
extracted with EtOAc (3 20 mL). The combined organic extracts were dried over Na2SO4,
×
filtered, and evaporated under reduced pressure. The product was purified by column
chromatography (EtOAc/n-hexane, 1/2, v/v). Yellow solid; yield: 64%; 1H-NMR (400 MHz,
CDCl3)
7.52–7.57 (m, 4H, Ar-C
1H, Ar- ), 8.11 (s, 1H, Ar-
δ
2.63 (s, 3H, Ar-CH3), 3.14 (s, 3H, SO2CH3), 7.44–7.48 (m, 1H, Ar-CHC
CHSO2CH3 and 3 Ar- ), 7.61–7.65 (m, 3H, Ar-
); 13C-NMR (100 MHz, CDCl3):
15.08, 42.96, 60.48, 65.58,
H
SO2CH3),
H
×
H
H), 7.87 (s,
H
H
δ
100.25, 116.60, 116.89, 121.68, 122.48, 124.96, 127.66 (2C), 128.31, 129.38 (2C), 130.69, 143.40,
149.56, 150.40, 157.12, 161.99; HRMS (ESI) m/z calculated for C21H17O5S [M + H]+ 381.0791,
found 381.0795.; Purity by HPLC (0–18 min; 70% n-hexane/isopropanol): 99%.
Synthesis of 6-(3-bromo-4,5-dihydroisoxazol-5-yl)-5-methyl-3-phenyl-7H-furo[3,2-g]chromen-
7-one (10)
To a cooled (
−
15 ◦C) solution of 5-methyl-3-phenyl-6-vinyl-7H-furo[3,2-g]chromen-7-
one ( ) (145 mg, 0.48 mmol, 1 equiv.) and 1,1-dibromoformaldoxime (148 mg, 0.73 mmol,
7
1.5 equiv.) in DMF (10 mL), an aqueous solution of NaHCO3 (1 mL, 112 mg, 1.3 mmol,
2.7 equiv.) was added. The reaction was then warmed to room temperature and stirred
for 5 h. Then, the solution was diluted with CH2Cl2 (20 mL) and washed with brine
(20 mL). The organic extract was dried over Na2SO4, filtered, and the solvents removed
under reduced pressure. The product was purified by column chromatography (EtOAc/n-
1
hexane, 1/4, v/v) to yield pale yellow solid. Yield: 76%. H-NMR (400 MHz, CDCl3)
δ
2.60 (s, 3H, Ar-CH3), 3.47 (dd, J = 17.1, 11.8 Hz, 1H, one H of CH2), 3.62 (dd, J = 17.1,
10.4 Hz, 1H, one H of CH2), 6.09 (dd, J = 11.8, 10.4 Hz, 1H, CH2C O), 7.42–7.48 (m, 2H,
), 7.83 (s, 1H, Ar- ), 8.06 (s, 1H,
15.27, 46.05, 77.81, 100.01, 116.61, 116.75, 119.73,
H
Ar-
Ar-
H
H
), 7.51–7.57 (m, 2H, Ar-
H
), 7.60–7.64 (m, 2H, Ar-
H
H
); 13C-NMR (101 MHz, CDCl3)
δ
122.36, 124.29, 127.60, 128.19, 129.33, 130.84, 137.27, 143.12, 150.88, 151.48, 157.17, 159.50;
HRMS (ESI) m/z calculated for C21H15O4NBr [M + H]+ 424.0179, found 424.0179. Purity by
HPLC (0–18 min; 70% n-hexane/isopropanol): 99%.
Synthesis of 5-methyl-3-phenyl-6-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ethyl)-
7H-furo[3,2-g]chromen-7-one (11):
5-Methyl-3-phenyl-6-vinyl-7H-furo[3,2-g]chromen-7-one (7) (0.09 mmol, 1 equiv.) was
dissolved in 1,4-dioxane (2 mL) and then bis(pinacolato)diboron (1,5 equiv.), cesium fluo-
ride (2,5 equiv.) and MeOH (5 equiv.) were added. The reaction proceeded at 100 ◦C for
12 h. The reaction mixture was then diluted with EtOAc (15 mL) and filtrated over silica.
The filtrate was evaporated under reduced pressure and the product purified from the
crude mixture by column chromatography (EtOAc/n-hexane, 1/9, gradient to 1/1, v/v)
1
to yield yellow solid. Yield: 33%. H-NMR (400 MHz, CD3OD):
δ
0.94 (t, J = 8.5 Hz, 2H,
3), 2.68 (t, J = 8.5 Hz, 2H, C 2), 7.30–7.35
), 7.62–7.62 (m, 2H, Ar- ), 7.99 (s, 1H, Ar- ), 8.05
); 13C-NMR (100 MHz, CDCl3):
14.12, 17.77, 23.59 (4C), 29.40, 83.06, 85.22
CH
2), 1.13 (s, 12H, C(C
(m, 1H, Ar- ), 7.41–7.47 (m, 3H, Ar-
(s, 1H, Ar-
H
3)2), 2.48 (s, 3H, Ar-C
H
H
H
H
H
H
H
δ
(2C), 87.21, 102.00, 102.50, 105.99, 127.09 (2C), 127.58, 127.64, 128.75 (2C), 136.06, 141.54,
146.52, 152.51, 157.66, 161.81; HRMS (ESI) m/z calculated for C26H28O5B [M + H]+ 431.2024,
found 431.2023.; Purity by HPLC (0–18 min; 95% n-hexane/isopropanol): 97%.
Synthesis of 3-(4-methyl-2-oxo-7-(2-oxo-2-phenylethoxy)-2H-chromen-3-yl)propanoic
acid (13):
To a stirred solution of 12 (788 mg, 2 mmol, 1 equiv.) in dioxane (20 mL), HCl (1 M,
20 mL, 10 equiv.) was added. The reaction mixture was heated at reflux temperature for 2 h.
Dioxane was then evaporated under reduced pressure, the precipitate that formed filtered
1
off and washed with H2O. Yield: 94%. H-NMR (400 MHz, CDCl3)
δ
2.44 (s, 3H, CH3),
2COOH), 5.38 (s, 2H,
), 7.49–7.60 (m, 3H,
Ar-H); HRMS (ESI) m/z
2.67 (t, J = 7.6 Hz, 2H, C
H
2CH2COOH), 2.96 (t, J = 7.6 Hz, 2H, CH2C
H2), 6.78 (d, J = 2.6 Hz, 1H, Ar- ), 6.95 (dd, J = 8.9, 2.6 Hz, 1H, Ar-H
H
C
H
3
×
Ar-
H
), 7.66 (t, J = 7.4 Hz, 1H, Ar-
H
), 7.82–8.07 (m, 2H, 2
×
calculated for C21H17O6 [M − H]− 365.1031, found 365.1030.
Synthesis of 3-(4-methyl-2-oxo-7-(2-oxo-2-phenylethoxy)-2H-chromen-3-yl)propanal (14):

Molecules 2021, 26, 356
13 of 18
To a suspension of 13 (366 mg, 1 mmol, 1 equiv.) in toluene (20 mL), dried over 3 Å
molecular sieves, a catalytic amount of anhydrous DMF (5 drops) and SOCl2 (218 µL,
357 mg, 3 mmol, 3 equiv.) were added under argon. The reaction mixture was stirred
at room temperature for 17 h and then the volatiles were evaporated to obtain a white
solid that was dried under vacuum for 15 min to remove SOCl2. Toluene (20 mL), dried
over 3 Å molecular sieves, was added to the dried solid (under argon), followed by the
addition of 10% Pd/BaSO4 (72 mg, 30% [w/w]). The reaction mixture was heated to
100 ◦C and stirred under a stream of hydrogen (1 atm) for 2 h. The reaction mixture was
then evaporated to dryness and the compound was purified by column chromatography
1
(EtOAc/n-hexane, 1/1, v/v, dry loading). White solid, yield: 59%. H-NMR (400 MHz,
CDCl3)
CH2CH2CHO), 5.38 (s, 2H, CH2), 6.78 (d, J = 2.6 Hz, 1H, Ar-
1H, Ar- ), 7.50–7.59 (m, 3H, 3 Ar- ), 7.66 (t, J = 7.4 Hz, 1H, Ar-H
δ
2.43 (s, 3H, CH3), 2.77 (t, J = 7.4 Hz, 2H, CH2CH2CHO), 2.94 (t, J = 7.4 Hz, 2H,
), 6.95 (dd, J = 8.9, 2.6 Hz,
), 7.04–8.06 (m, 2H,
O); HRMS (ESI) m/z calculated for C21H19O5
H
H
×
H
2 × Ar-H), 9.83 (t, J = 1.0 Hz, 1H, CH2CH2C
H
[M + H]+ 351.1227, found 351.1221.
Synthesis of 3-(5-methyl-7-oxo-3-phenyl-7H-furo[3,2-g]chromen-6-yl)propanal (15):
To a suspension of 14 (519 mg, 1.48 mmol, 1 equiv.) in propan-2-ol (35 mL), an aqueous
solution of NaOH (14.8 mL, 1 M, 10 equiv.) was added. The reaction mixture was stirred
at 60 ◦C for 15 min. After the reaction was complete (monitored by TLC), the resulting
red solution was acidified with 1 M HCl (15 mL) to get a yellow precipitate. The reaction
mixture was evaporated under reduced pressure. H2O (50 mL) was added to the dry
residue, which was extracted with CH2Cl2 (2
washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and evaporated under
×
50 mL), the combined organic extracts were
reduced pressure. The compound was purified by column chromatography (EtOAc/n-
1
hexane, 1/2, v/v, dry loading). White solid, yield: 61%. H-NMR (400 MHz, CDCl3)
δ
2.55
2CHO),
), 7.60–7.67 (m, 2H,
), 9.86 (t, J = 1.0 Hz, 1H, CH2CH2C O);
15.67, 20.85, 42.53, 99.97, 115.99, 117.34, 122.46, 122.98, 124.09,
(s, 3H, C
7.47–7.41 (m, 1H, Ar-
Ar- ), 7.83 (s, 1H, Ar-
13C-NMR (101 MHz, CDCl3)
H
3), 2.82 (t, J = 7.4 Hz, 2H, C
), 7.49 (s, 1H, Ar-
), 8.00 (s, 1H, Ar-
H
2CH2CHO), 3.01 (t, J = 7.4 Hz, 2H, CH2C
H
H
H
), 7.50–7.57 (m, 2H, 2 Ar-
×
H
2
×
H
H
H
H
δ
127.72, 128.18, 129.39, 131.24, 142.94, 147.70, 150.56, 156.58, 161.72, 201.40; HRMS (ESI) m/z
calculated for C21H17O4 [M + H]+ 333.1121, found 333.1116. Purity by HPLC (0–18 min;
70% n-hexane/isopropanol): 87%.
Synthesis of 3-(5-methyl-7-oxo-3-phenyl-7H-furo[3,2-g]chromen-6-yl)acrylaldehyde (16):
To a solution of the aldehyde 15 (53 mg, 0.16 mmol, 1 equiv.) in a mixture of diox-
ane (1.5 mL) and H2O (20
µ
L), SeO2 (35 mg, 0.32 mmol, 2 equiv.) was added and
◦
the reaction mixture was irradiated in a microwave reactor at 150 C (250 W) for 1 h.
The reaction mixture was then evaporated to dryness and the compound was purified by
column chromatography (EtOAc/n-hexane, 1/2, v/v, dry loading). White solid, yield:
10%. 1H-NMR (400 MHz, CDCl3)
CHC CHO), 7.46 (t, J = 7.4 Hz, 1H, Ar-
7.61–7.69 (m, 3H, 2 Ar- and C CHCHO), 7.85 (s, 1H, Ar-
(d, J = 7.5 Hz, 1H, CHCHC
O); 13C-NMR (101 MHz, CDCl3)
δ
2.74 (s, 3H, CH3), 7.32 (dd, J = 15.8, 7.5 Hz, 1H,
), 7.51 (s, 1H, Ar-
), 7.52–7.59 (m, 2H, 2 × Ar-H),
), 8.14 (s, 1H, Ar- ), 9.74
16.21, 100.29, 116.93,
H
H
H
×
H
H
H
H
H
δ
117.39, 118.27, 122.59, 124.84, 127.79, 128.42, 129.49, 130.86, 134.87, 143.45, 143.55, 151.20,
152.79, 157.88, 158.95, 194.53; HRMS (ESI) m/z calculated for C21H15O4 [M + H]+ 331.0965,
found 331.0979. Purity by HPLC (0–18 min; 70% n-hexane/isopropanol): 97%.
Synthesis of 5-methyl-N-((S)-1-((R)-2-methyloxiran-2-yl)-L-oxopropan-2-yl)-7-oxo-3-
phenyl-7H-furo[3,2-g]chromene-6-carboxamide (20):
To a cooled (0 ◦C) solution of compound 17 (160 mg, 0.50 mmol, 1 equiv.) in DMF
(4 mL), HATU (285 mg, 0.75 mmol, 1.5 equiv.) and HOBt hydrate (115 mg, 0.75 mmol,
1.5 equiv.) were added. In a separate round-bottom flask, compound 18 (115 mg, 0.50 mmol,
◦
1 equiv.) was dissolved in CH2Cl2 (3 mL) at 0 C, followed by the addition of TFA
◦
(3 mL). After 30 min of stirring at 0 C, the volatiles were evaporated under reduced
pressure thoroughly, the residue was dissolved in CH2Cl2 and slowly added to the mixture
containing compound 17 at 0 ◦C. After 5 min, DIPEA (348
µL, 285 mg, 2.0 mmol, 4 equiv.)

Molecules 2021, 26, 356
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was added and the reaction mixture stirred at room temperature for 24 h. Then, the solvent
was evaporated and the product purified by column chromatography (EtOAc/n-hexane,
1
1/1, v/v, dry loading) without additional work-up. Off-white solid, yield: 16%. H-NMR
(400 MHz, CDCl3)
H3), 2.96 (d, J = 5.0 Hz, 1H, one H of oxirane CH2), 3.39 (app d, J = 5.0 Hz, 1H, one H
of oxirane CH2), 4.72 (p, J = 6.7 Hz, 1H, C CH3), 7.42–7.48 (m, 1H, Ar- ), 7.51 (s, 1H,
Ar- ), 7.52–7.57 (m, 2H, Ar- ), 7.59–7.65 (m, 3H, CON + Ar- ), 7.85 (s, 1H, Ar- ), 8.13
(s, 1H, Ar- 16.87, 16.92 (2C), 48.68, 52.70, 59.15, 100.04,
), 13C-NMR (101 MHz, CDCl3)
δ 1.44 (d, J = 7.0 Hz, 3H, CHCH3), 1.57 (s, 3H, CH3), 2.75 (s, 3H, Ar-
C
H
H
H
H
H
H
H
H
δ
116.76, 117.32, 118.53, 122.43, 124.69, 127.63, 128.23, 129.33, 130.71, 143.27, 150.83, 155.64,
157.64, 160.02, 163.86, 208.01; HRMS (ESI) m/z calculated for C25H22O6N [M + H]+ 432.1442,
found 432.1438. Purity by HPLC (0–18 min; 70% n-hexane/isopropanol): 96%.
Synthesis of 5-methyl-N-((S)-1-((R)-2-methyloxiran-2-yl)-L-oxo-3-phenylpropan-2-yl)-
7-oxo-3-phenyl-7H-furo[3,2-g]chromene-6-carboxamide (21):
To a cooled (0 ◦C) solution of compound 17 (28 mg, 0.087 mmol, 1 equiv.) in CH2Cl2
(4 mL), HATU (40 mg, 0.11 mmol, 1.2 equiv.) and HOBt hydrate (17 mg, 0.11 mmol,
1.2 equiv.) were added. In a separate round-bottom flask, compound 19 (27 mg, 0.087 mmol,
1 equiv.) was dissolved in CH2Cl2 (2 mL) at 0 ◦C, followed by the addition of TFA (2 mL).
◦
After 30 min of stirring at 0 C, the volatiles were evaporated under reduced pressure thor-
oughly, the residue was dissolved in CH2Cl2 and slowly added to the mixture containing
compound 17 at 0 ◦C. After 5 min, DIPEA (58
µL, 45 mg, 0.35 mmol, 4 equiv.) was added
and the reaction mixture stirred at room temperature for 24 h. Then, the solvent was evap-
orated and the product purified by column chromatography (EtOAc/n-hexane, 1/2, v/v,
1
dry loading) without additional work-up. Off-white solid, yield: 11%. H-NMR (400 MHz,
CDCl3)
δ 1.55 (s, 3H, CH3), 2.65 (s, 3H, Ar-CH3), 2.88 (dd, J = 13.7, 8.7 Hz, 1H, one H of
oxirane CH2), 2.98 (d, J = 5.0 Hz, 1H, one H of oxirane CH2), 3.27 (dd, J = 13.7, 4.8 Hz,
1H, one H of CHCH2Ph), 3.47 (dd, J = 5.0, 0.5 Hz, 1H, one H of CHCH2Ph), 4.99 (symm
m, 1H, C
Ar- ), 7.52–7.56 (m, 2H, Ar-
J = 6.7 Hz, 1H, CON ), 8.11 (s, 1H, Ar-
H
CH3), 7.27–7.35 (m, 5H, Ar-
), 7.59–7.63 (m, 2H, Ar-
); 13C-NMR was not recorded due to insufficient
H
), 7.42–7.47 (m, 1H, Ar-
H
), 7.50 (d, J = 0.4 Hz, 1H,
H
H
H), 7.84 (s, 1H, Ar-
H), 7.85 (br d,
H
H
amount of the final product; HRMS (ESI) m/z calculated for C31H26O6N [M + H]+ 508.1755,
found 508.1755. Purity by HPLC (0–18 min; 95% n-hexane/isopropanol): 97%.
Synthesis of 3-bromo-N-((5-methyl-7-oxo-3-phenyl-7H-furo[3,2-g]chromen-6-yl)methyl)
propanamide (23):
To a cooled (0 ◦C) solution of compound 22 (92 mg, 0.3 mmol, 1 equiv.) in CH2Cl2
(10 mL), K2CO3 (50 mg, 0.36 mmol, 1.2 equiv.) was added. After 5 min, 3-bromopropanoyl
chloride (36
mixture was then stirred at room temperature for 3 h. The reaction was quenched by
the addition of H2O (20 mL) and the mixture was extracted with EtOAc (3 50 mL).
µ
L, 62 mg, 0.36 mmol, 1.2 equiv.) was added drop-wise at 0 ◦C. The reaction
×
The combined organic extracts were dried over Na2SO4, filtered, and evaporated under
reduced pressure. The product was purified by column chromatography (EtOAc/n-hexane,
1/2, v/v). White solid, yield: 90%. 1H-NMR (400 MHz, CDCl3)
δ
2.73 (s, 3H, CH3),
2,74 (t, J = 6.7 Hz, 2H, COCH2), 3.61 (t, J = 6.7 Hz, 2H, CH2Br), 4.52 (d, J = 6.2 Hz, 2H,
Ar-CH2NH), 7.42–7.47 (m, 1H, Ar- ), 7.51–7.56 (m, 3H, Ar- ), 7.61–7.64 (m, 2H, Ar-
), 7.83 (s, 1H, Ar- ), 8.07 (s, 1H, Ar- 15.59, 27.13,
); 13C-NMR (101 MHz, CDCl3)
H
H
H
H
H
δ
36.69, 39.51, 100.01, 116.75, 117.07, 120.68, 122.43, 124.31, 127.61 (2C), 128.15, 129.32 (2C),
130.96, 142.99, 149.61, 150.66, 156.81, 162.37, 169.52; HRMS ( m/z ) (ESI): calculated for
C22H19O4NBr [M + H]+ 440.0492, found: 440.0490; Purity by HPLC (0–18 min; 70% n-
hexane/isopropanol): 96%.
Synthesis of 1-((5-methyl-7-oxo-3-phenyl-7H-furo[3,2-g]chromen-6-yl)methyl)azetidin-
2-one (24):
◦
To a cooled (0 C) solution of compound 23 (66 mg, 0.15 mmol, 1 equiv.) in DMF
(15 mL), NaOtBu (16 mg, 0.17 mmol, 1.1 equiv.) was added. The reaction mixture was
stirred at room temperature for 24 h. The reaction was quenched by the addition of
H2O (20 mL) and the mixture was extracted with EtOAc (3
×
50 mL). The combined

Molecules 2021, 26, 356
15 of 18
organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pres-
sure. The product was purified by column chromatography (EtOAc/n-hexane, 1/2, v/v).
1
White solid, yield: 87%. H-NMR (400 MHz, CDCl3)
δ 2.73 (s, 3H, CH3), 2.74 (t, J = 6.4 Hz,
2H, azetidin-2-one-CH2), 3.61 (t, J = 6.4 Hz, 2H, azetidin-2-one-CH2), 4.52 (s, 2H, Ar-
H2N), 7.42–7.47 (m, 1H, Ar- ), 7.52 (s, 1H, Ar- ), 7.53–7.56 (m, 2H, Ar- ), 7.61–7.65
(m, 2H, Ar-H), 7.84 (s, 1H, Ar- ), 8.07 (s, 1H, Ar- 29.72,
); 13C-NMR (101 MHz, CDCl3)
C
H
H
H
H
H
δ
31.95, 37.04, 38.55, 102.72, 112.28, 115.88, 116.54, 116.80, 122.43, 122.51, 127.62 (2C), 128.16,
128.74, 129.33 (2C), 132.78, 143.00, 156.80, 157.68, 178.20; HRMS ( m/z ) (ESI): calculated
for C22H18O4N [M + H]+ 360.1230, found: 360.1222; Purity by HPLC (0–18 min; 70%
n-hexane/isopropanol): 87%.
3.3. Residual Activity Measurements
The screening of compounds was performed at 1
buffer (0.01% SDS, 50 mM Tris-HCl, 0.5 mM EDTA, pH 7.4). Stock solutions of compounds
were prepared in DMSO. To 50 L of each compound, 25 L of 0.8 nM human iCP or human
cCP (both from Boston Biochem, Inc., Cambridge, MA, USA) was added. After 30 min
incubation at 37 ◦C, the reaction was initiated by the addition of 25
L of 100 M relevant
fluorogenic substrate: acetyl-Nle-Pro-Nle-Asp-AMC (Ac-nLPnLD-AMC, [Bachem, Buben-
dorf, Switzerland]) for 1, acetyl-Pro-Ala-Leu-7-amino-4-methylcoumarin (Ac-PAL-AMC,
[Boston Biochem, Inc., Cambridge, MA, USA]) for 1i, t-butyloxycarbonyl-Leu-Arg-Arg-
7-amino-4-methylcoumarin (Boc-LRR-AMC, [Bachem, Bubendorf, Switzerland]) for
and 2i, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC [Bachem,
Bubendorf, Switzerland]) for 5 and 5i. The reaction progress was recorded on the
µM final concentrations in the assay
µ
µ
µ
µ
β
β
β2
β
β
β
BioTek Synergy HT microplate reader by monitoring fluorescence at 460 nm (λex = 360 nm)
◦
for 90 min at 37 C. The initial linear ranges were used to calculate the velocity and to
determine the residual activity.
In the case of the β1, β1i, β2, and β2i activity inhibition determination, the assay
buffer was modified; SDS was replaced with the proteasomal activator PA28
Biochem, Inc., Cambridge, MA, USA).
α (Boston
3.4. Molecular Modelling
Compounds were prepared for docking using LigPrep (Schrödinger Suite 2020-2,
Schrödinger, LLC, New York, NY, USA, 2020) to account for all possible tautomers and
ionization states at pH 7.0
proteasome with human 5i and
was used for docking. The binding site is defined by the chain K (
1), so all other chains were removed. Protein Preparation Wizard [50] was used to add
±
2.0. The X-ray structure (PDB: 5M2B, [43]) of yeast 20S
1 subunits in complex with noncovalent inhibitor Ro19
5i) and neighbouring L
β
β
β
(β
hydrogen atoms, protonate residues at pH 7, refine the H-bond network and to perform a
restrained minimization. The receptor’s grid box required for docking calculations was
centred on the corresponding co-crystallized ligand. Noncovalent docking was performed
using Glide [51], with the following parameters: XP (extra precision), flexible ligand
sampling, perform postdocking minimization. Covalent docking was performed with
CovDock program [52] using the pose prediction mode with default setup and Thr1 defined
as the reactive residue. Nucleophilic addition to a double bond (oxathiazolone) was selected
as the reaction.
4. Conclusions
Here, we showed that the introduction of 12 new electrophilic warheads at position
3 of the psoralen ring led to compounds with abrogated inhibition of the iCP (especially
β5i subunit). As already described in the Introduction, it is imperative that the initial non-
covalent binding of a given compound is followed by the positioning of the electrophilic
‘warhead’ near the desired nucleophilic amino-acid residue of the protein to achieve
covalent interaction. Poor inhibition results were in our cases most probably due to
the mispositioning of the electrophilic carbon and the catalytic Thr1Oγ (Figure 7). The

Molecules 2021, 26, 356
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oxathiazolone thus remains the optimal electrophilic moiety for this compound class.
Despite somewhat disappointing results, the obtained data will help steer our future
research in the field of psoralen-based iCP inhibitors, e.g., when designing inhibitors which
simultaneously inhibit two iCP subunits as it was established that simultaneous inhibition
of β1i and β5i is necessary to achieve significant anti-inflammatory effects.
Author Contributions: Conceptualization, J.M., S.G., and I.S.; formal analysis, M.G. and I.S.; fund-
ing acquisition, S.G.; investigation, E.S.S., M.P., L.R., and A.Š.; methodology, E.S.S., M.P., M.G.,
and I.S.; project administration, S.G.; supervision, J.M. and I.S.; writing—original draft, E.S.S.;
writing—review & editing, S.G. and I.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Slovenian Research Agency, research core funding
No. P1-0208, Grant number N1-0068 to S.G., and Grant number J3-1745 to M.G.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors acknowledge Maja Frelih for HRMS measurements.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of all compounds, except compound 21 are available from the authors.
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