New C4- and C1-derivatives of furo[3,4-c]pyridine-3- ones and related compounds: Evidence for site-specific inhibition of the constitutive proteasome and its immunoisoform

Article abstract of DOI:10.1016/j.bmcl.2014.01.072

A set of 18 new C⁴ and C¹ derivatives of nor-cerpegin (1,1-dimethyl furo[3,4-c]pyridine-3-one), 6 model compounds (γ- and δ-lactones) and 20 furo- or thieno[2,3-d]-pyrimidine-4-one related compounds were designed and synthesized. Each compound was assayed for inhibition of CT-L, T-L and PA proteolytic activities of 20S constitutive proteasome (c20S). Most performant compounds were also assayed on 20S immunoproteasome (i20S). Compound 10 with a benzylamino group at C⁴ and dimethylated at C¹ of the furopyridine ring was the most efficient PA site-specific inhibitor of the c20S (IC50cPA of 600 nM) without noticeable inhibition of the i20S PA site (iPA). In silico docking assays for 10 at the iPA catalytic site revealed the absence of poses normally observed for this compound and related ones at the constitutive PA site (cPA). The thieno[2,3-d]pyrimidine-4-one 40 was T-L site-specific with a mild inhibition of both c20S and i20S in vitro (IC50cT-L of 9.9 μM and IC50iT-L of 6.7 μM). In silico docking assays of 40 at T-L sites of c20S and i20S revealed almost identical first rank poses in the two types of sites with no possibility left for nucleophilic attack by Thr1 as observed for the fused furopyridine-3-one 10.

Full text of DOI:10.1016/j.bmcl.2014.01.072

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1571–1580
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
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New C4- and C1-derivatives of furo[3,4-c]pyridine-3-ones and related
compounds: Evidence for site-specific inhibition of the constitutive
proteasome and its immunoisoform
Anna Hovhannisyan c, , The Hien Pham a,b, , Dominique Bouvier d, , Alexander Piroyan c, Laure Dufau a,b
,
Lixian Qin a,b, Yan Cheng a,b, Gagik Melikyan c, Michèle Reboud-Ravaux a,b, Michelle Bouvier-Durand a,b,
⇑
a Sorbonne Universités, UPMC Univ Paris 06, UMR 8256, ERL U1164, B2A, Biological Adaptation and Ageing, Integrated Cellular Ageing and Inflammation, Molecular &
Functional Enzymology, Case 256, 7 Quai St Bernard, F-75005 Paris, France
b CNRS, UMR 8256, B2A, Biological Adaptation and Ageing, F-75005 Paris, France
c Department of Organic Chemistry, Yerevan State University, A. Manoogian Str. 1, 0025 Yerevan, Armenia
d Sorbonne Universités, UPMC Univ Paris 06, Atelier de Bioinformatique, Case courrier 1202, 4 Place Jussieu, F 75252 Paris Cedex 05, France
a r t i c l e i n f o
a b s t r a c t
A set of 18 new C4 and C1 derivatives of nor-cerpegin (1,1-dimethyl furo[3,4-c]pyridine-3-one), 6 model
compounds ( - and d-lactones) and 20 furo- or thieno[2,3-d]-pyrimidine-4-one related compounds were
designed and synthesized. Each compound was assayed for inhibition of CT-L, T-L and PA proteolytic
Article history:
Received 25 November 2013
Revised 22 January 2014
Accepted 23 January 2014
Available online 3 February 2014
c
activities of 20S constitutive proteasome (c20S). Most performant compounds were also assayed on
20S immunoproteasome (i20S). Compound 10 with a benzylamino group at C4 and dimethylated at C1
cPA
50
of the furopyridine ring was the most efficient PA site-specific inhibitor of the c20S (IC
of 600 nM)
Keywords:
without noticeable inhibition of the i20S PA site (iPA). In silico docking assays for 10 at the iPA catalytic
site revealed the absence of poses normally observed for this compound and related ones at the consti-
tutive PA site (cPA). The thieno[2,3-d]pyrimidine-4-one 40 was T-L site-specific with a mild inhibition of
Furo[3,4-c]pyridine-3-ones
Furo- and thieno[2,3-d]pyrimidine-4-ones
CT-L
T-L and PA proteolytic activities
Constitutive c20S proteasome
Immunoproteasome i20S
In silico docking
cT-L
50
iT-L
50
both c20S and i20S in vitro (IC
of 9.9
l
M and IC of 6.7 lM). In silico docking assays of 40 at T-L sites
of c20S and i20S revealed almost identical first rank poses in the two types of sites with no possibility left
for nucleophilic attack by Thr1 as observed for the fused furopyridine-3-one 10.
Ó 2014 Elsevier Ltd. All rights reserved.
In
1,1,5-trimethylfuro[3,4-c]pyridine-3,4-dione (cerpegin, a pyridi-
none-fused -lactone) and their use as inhibitors of 20S protea-
some, with initial optimization when compared to the series
originally described.2 Our present work takes advantage of inter-
esting properties of the identified lead compounds as selective
inhibitors of the post-acid activity of mammalian constitutive
20S proteasomes. A search for new analogs with optimized
a
preceding article,1 we reported on derivatives of
inhibitory power and preserved selectivity was conducted. This
work also takes advantage of the knowledge of several classes of
inhibitors of the catalytic core (20S or CP) already described for
the constitutive 26S proteasome3–6 and more recently, for the
immunoproteasome (reviewed in Ref. 7) (c20S and i20S,
respectively).
In eukaryotic cells, 26S proteasomes are essential proteolytic
components of the general ubiquitin-proteasome pathway (UPS),
with a dependency for energy and ubiquitin.3–6 Many cellular
functions (cell cycle progression,8 apoptosis,9 transcription,10
checkpoint control of DNA damage and DNA repair,10 degradation
of abnormal proteins by the ERAD (Endoplasmic Reticulum-
Associated Degradation)11 pathway, are dependent on the
proteasomal action to specifically cleave key protein substrates
into small peptides. In mammalian lymphocytes and monocytes,
c20S also shapes a part of the peptides loaded on the major
histocompatibility complex class I (MHC-I).3–5,7 In all mammalian
cells, another panel of MHC-I peptides is shaped by the immuno-
proteasome after induction by cytokines.12
c
Abbreviations: DMF DMA, dimethylformamide dimethylacetal; DMSO, dimeth-
ylsulfoxide; c20S, 20S constitutive proteasome catalytic core; i20S, immunopro-
teasome catalytic core; CT-L, chymotrypsin-like; T-L, trypsin-like; PA, post-acid or
caspase-like; AMC, 7-amino-4-methyl-coumarin; b-NA, b-naphtylamide; CPK,
Corey Pauling Koltun; PDB, protein data bank.
⇑
Corresponding author. Tel.: +33 1 44 27 59 62; fax: +33 1 44 27 51 40.
E-mail addresses: annahovh@gmail.com (A. Hovhannisyan), bsthehien@yahoo.
com (T.H. Pham), dbouvier@snv.jussieu.fr (D. Bouvier), aleksandr.piroyan@gmail.
com (A. Piroyan), dufaulaure@yahoo.fr (L. Dufau), lixian.qin@free.fr (L. Qin),
cyan86520@hotmail.com (Y. Cheng), acect@yahoo.com (G. Melikyan), Michele.
Reboud@upmc.fr (M. Reboud-Ravaux), mbouvier@snv.jussieu.fr (M. Bouvier-Durand).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.01.072
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1572
A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
The CP particles of c20S and i20S form a similar cylindrical
structure, each comprising four stacked heptameric rings ( 1–7
b1–7 b1–7 1–7). The different proteolytic activities of c20S or
investigated by in silico docking. Previous studies from our group
have suggested a covalent mechanism of inhibition associated with
a
a
a unique mode of binding of cerpegin-derived c-lactones to the bo-
i20S are confined to the two identical inner b rings. In a single b
ring, only the b1, b2 and b5 subunits possess a proteolytic activity,
the so-called caspase-like (C-L or PA), trypsin-like (T-L) and chy-
motrypsin-like (CT-L) activity, respectively.3–5,13–17 Whatever the
nature of the catalytic particle (c20S or i20S), each type of proteo-
lytic site possesses a threonine residue at position 1 capable of
nucleophilic attack on the substrates. However, specificity of the
respective sites (b1, b2 and b5) can be distinguished in vitro by
placing acidic, basic or hydrophobic residues at the C-terminal
end of fluorogenic substrates. Despite small variations in the amino
acid sequences of the respective catalytic subunits of i20S, b1i
(LMP2), b2i (MECL-1) and b5i (LMP7), the same substrates can be
used as those for c20S to evaluate the respective activities. b5i sub-
units have been recently reported as key regulators of cytokine
production with selective inhibition by the epoxyketone PR-957
and therapeutic applications for experimental arthritis.18 A recent
X-ray study7 of this compound bound to the mouse i20S or c20S
crystal structures showed the absence of conformational changes
in the S1 specificity pocket of b5i site, explaining the selectivity
of PR-957 for b5i. Site-specific proteasome inhibitors are not con-
fined to b5 sites since b1 ones have been shown to be the specific
targets of IPSI-00119 and LMP2-ek19 in the case of i20S. Both b1c
and b1i sites are inhibited by peptido-epoxyketones YU-102,20
NC-00121 and LU-102.22 As regards b2 sites, NC-022 is a specific
inhibitor of T-L activity.23
vine b1c active site.1,2
The general structures of the molecules synthesized and evalu-
ated in this study are presented in Figure 1(B–E) whereas reference
compounds are presented in Figure 1A. The b-lactone of SalA
(Fig. 1A, top) is attacked by the nucleophilic Thr1 and this leads
to formation of a highly stable acyl-enzyme.24 The
c-lactone ring
of cerpegin (Fig. 1A, bottom) is also susceptible to nucleophilic at-
tack by Thr1 and we have recently reported kinetic results showing
covalent proteasome inhibition by a cerpegin derivative.2 Further-
more, cerpegin derivatives substituted at N5 or at N5 and C1
showed selectivity towards the post-acid activity (IC50 2–
10
been designed to retain the
l
M).1,2 Based on these results, new series of molecules have
c
-lactone of the fused furopyridine ring
of cerpegin. C4- and C1-substituted furo[3,4-c]pyridine-3-one
derivatives with main variations of the functional groups at C4 po-
sition (labeled R in Fig. 1B) were synthesized. These R groups were
mainly chosen on the basis of our previous results where they
appeared as efficient N5 substituents.1,2
Some other
c-lactones not fused to a pyridine ring were also
designed and prepared (Fig. 1C, 19–22), together with two pyri-
dine-fused d-lactones (Fig. 1C, 23 and 24) as model compounds
to evaluate the effects of the lactone environment.
The role of carbonyl-derived chemical groups other than
c-lactones, placed in various heterocyclic environments (fused
furo- and thienopyrimidine-4-ones), was also investigated
Cell cycle progression and apoptosis induction are the cellular
functions best known for their susceptibility to proteasome inhibi-
tion. The dipeptide boronate bortezomib (or VelcadeÒ, initially
PS341) was the only one that came to therapeutic step among
the first generation of inhibitors. Successful treatments with this
molecule were reported in 2003 on multiple myeloma and then
in 2006 on relapsed mantle cell lymphoma. Because of drastic side
effects and the appearance of resistant cells, a second generation of
inhibitors has been developed. Two other peptide-boronates, CEP-
18770 and MLN9708, the tetrapeptide epoxyketone carfilzomib
and the b-lactone salinosporamide A, have been the subject of
intensive clinical studies.24 Carfilzomib (Kyprolis or PR-171) has
been recently approved by FDA for the treatment of multiple
myeloma.25 Salinosporamide A (SalA, NPI 0052 or marizomib) be-
longs to a large family of natural compounds (omuralide, b-lactosin
A, reviewed in Ref. 6) and is a highly potent inhibitor of c20S with a
b-lactone working as a pharmacophore. SalA has been retained in
this study as a reference b-lactone24 as well as cerpegin derivatives
(Fig. 1D and E).
The aforementioned C4- and C1-substituted furo[3,4-c]pyridine-
3-one derivatives (Fig. 1B) were synthesized as outlined in
Scheme 1A and B. Scheme 1A sums up steps for the synthesis of
compounds of series IV and VIII (1–4), III and VI (7–18). Com-
pounds XI (5, 6) were synthesized according to Scheme 1B. The un-
fused
c-lactone models 19, 20, 21, 22 were synthesized as
previously reported (19, 20,35,36 21,2 2237). The pyridinone-fused
d-lactones 23 and 24 were prepared as mentioned in38 for 23,
and according to Scheme 1C for 24.
The most convenient way of synthesis of nor-cerpegin (1,1-di-
methyl furo[3,4-c]pyridine-3,4-dione) (compound 1 in series IV)
was based on the use of easily accessible carbonitriles I reacting
with DMF DMA (Scheme 1A, i). Further cyclization in a AcOH/HCl
(3:1) mixture of dimethylaminovinyl derivatives II led to high
yields of product IV (1) (Scheme 1A, iii). The same steps were used
to produce compound 2.
Primary 4-amino derivatives III (7–9) were obtained in high
yields by reacting intermediates II in 20% ammonia (Scheme 1A,
ii). For the synthesis of 4-substituted amino furo pyridines VI
(10–14), the 4-chloro-derivative V obtained from compounds IV
(7) and PCl5 interaction at 130–150 °C during 15–20 min was used
possessing a c
-lactone moiety.1,2 These two categories of lactones
are covalent inhibitors and, together with peptide mimetics, they
represent a large class of molecules known to target the 20S cata-
lytic core.26–28 Non-covalent inhibitors have been more recently
developed28 including hydroxyurea (HU) compounds,29 oxadiaz-
oles,30 and peptidic derivatives.31–34
(Scheme 1A, i
amines were reacted with V under reflux in xylene (Scheme 1A,
a), whereas secondary amines were reacted in boiling DMF
v). On a first way to compounds VI (10–14), primary
v
,
The aim of the present study was to synthesize new molecules
bearing a
c
-lactone moiety on the model of cerpegin and deriva-
(Scheme 1A,
(Scheme 1A,
v
, b), and aromatic amines were reacted in AcOH
tives1,2,35 with substitutions at the C4 of the furo[3,4-c]pyridine-
3-one skeleton. Optimization of their inhibitory power towards
c20S fractions in vitro was first investigated and the most interest-
ing compounds from this approach were selected for testing their
efficiency on i20S fractions. Furthermore, calpain 1 and cathepsin
B activities were assayed in order to check the specificity of protea-
some inhibition. Whole cell assays were also conducted to measure
intracellular proteasome inhibition and to evaluate cytotoxity.
New furo- and thieno[2,3-d]pyrimidine-4-one compounds were
also synthesized and assayed for their aptitude to inhibit c20S
proteasome. For the most potent compounds identified as inhibi-
tors of c20S or i20S, the possible mechanisms of binding were
v, c). All reactants are summed up in Supplementary
Table S1. On a second way, several substituted amino derivatives
VI (15–18) with a side-chain hydroxyl group were synthesized
from the primary amine III (7) through reaction with oxiranes
(Scheme 1A, vi). The presence of a hydroxyl group on a side-chain
could lead to significant improvement of the binding affinity to
proteasome active sites, as already reported in our investigations
on cerpegin N5 derivatives.2
For comparison purposes, 4-thioxo VIII (3, 4) (Scheme 1A) and
4-methyl XI (5, 6) (Scheme 1B) derivatives were synthesized. The
4-thioxo derivatives VIII (3, 4) were obtained from the correspond-
ing thiocarboxamides VII (Scheme 1A, i0) without isolating

A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
1573
4
1
C
and C derivatives of nor-cerpegin
Reference compounds
C
A
B
Other lactone compounds
and related furopyridines
1-18
19-22
23
24
NH
N
1= nor-cerpegin with R = OH
R2
O
R
R1
1
2
4
O
R , R = CH3, CH3
R1
R2
3
4
5
O
O
1
O
24
SalA
O
O
R = SH or CH3 or NHR’ with
Cerpegin2
R’= H, CH2 C6H5 or NHC(S)NH2
or (CH2)5 or C6H4pCl or C6H4SO2NH2 or
CH2CH(OH)CH2OC6H5 or CH2CH(OH)CH2OC6H4pCH3
or CH2CH(OH)CH2OC6H4pF or
R
N5
1 = C6H5 or CH=CHN(CH3)2
or CH=CHC6H5
1
2
R
R, R ,R = CH3
O
O
R1
R2
HN
1
O
2
5
1
R = C(S)NH2 or
O
N -Cerpegin derivative:
CH2CH(OH)CH2OC6H4pCl
O
O
CH(CH3)NH2
or COOC2H5 or
C(O)NH2
R= C6H4SO2NH2
1
2
1
2
R , R = CH3, CH3
or CH3, C2H5 or spiro-fused R /R =(CH2)5
R , R = CH3
1
2
Fused furopyrimidine-4-ones
Fused thienopyrimidine-4-ones
D
E
R1
O
R3
27
25, 26, 29-34
4
5
3
N
O
N
R2
O
6
4
R3
NH
S
N
5
R1
R2
N
3
O
R2
35-39
40-41
2
6 O
N
1
2
1
1
2
R , R = CH3,CH3
R =C6H5 , R = H
Fused R , R = CH3
R
3 = (CH2)2 C6H5
R
3 = (CH2)2 C6H4pCl or
CH2CHOHCH2N(C2H5)2
or (CH2)2 C6H4 pCl
or (CH2)2 C6H4 pF
or (CH2)3 C3H3N2,
or (CH2)3 C4H8NO
1
2
R , R = CH3, CH3
R
3 = H or C6H4Br
28
or CH2CH2OH
or (CH2)3 C3H3N2,
O
or (CH2)3 C4H8NO
42-44
2
N
or CH2CH(OH) CH2C4H8NO
or CH2CH(OH)CH2OC6H5
1
R =H, R = C6H4 Cl
O
N
R
3 = (CH2)2 C6H5 or
(CH2)2 C6H4pCl or
(CH2)2 C6H4 pF
Spiro-fused R , R2 = (CH2)5
1
1
2
R , R = C6H5, CH3
26
3
R
= H
Figure 1. General presentation of the C4 and C1 derivatives of nor-cerpegin, of related furopyridines and of fused furo- and thienopyrimidine-4-ones synthesized in this
study: (A) General structures of reference compounds are shown: SalA molecule with a b-lactone24 and cerpegin2 or a N5 cerpegin derivative (compound 1 in Ref. 1) with a
lactone moiety. (B) C4 and C1 derivatives of nor-cerpegin (1, 2 and 10–18, series IV, VI) and related furopyridines (8, 9, 3, 4 and 5, 6, series III, VIII and XI); R1 and R2
substituents for nor-cerpegin (1) are both a methyl group. (C) Other lactones models: -lactones (19–22) and d-lactones (23 and 24). (D) Fused furopyrimidine-4-ones: the
c
-
c
furan ring is now fused to a pyrimidine ring and the c
-lactone moiety absent but a carbonyl group is present at the C4 position of the pyrimidine ring (25–34). (E) Fused
thienopyrimidine-4-ones: the furan ring is now replaced by a thieno ring fused to a pyrimidine ring. A carbonyl group is present at the C4 position of the pyrimidine ring (35–
44).
intermediate dimethylaminovinyl derivatives. The initial
thiocarboxamides36 VII were obtained from lactones I. The
4-methyl-derivatives XI (5, 6) (Scheme 1B) were obtained from
3-acetyl-furan-2(5H)-one compounds IX through the correspond-
ing dimethylaminovinyl intermediates X by ring formation in the
presence of ammonia.
For each of these compounds, yields and melting points as well
as 1H NMR data are reported in Supplementary section. 13CNMR
and GCMS data are also reported for compounds 3, 7, 8, 10, 15,
18, 24 and 40.
Tables 1 and 2 show the IC50 values (concentrations giving 50%
inhibition) obtained for each kind of peptidase activity, after incu-
bation of a purified c20S fraction with the respective furo-pyri-
dine-3-ones and furo- or thienopyrimidine-4-ones. Assays were
conducted following the protocols previously described for each
activity1,2 and summarized in Ref. 39. A similar protocol was used
for the i20S purified fraction.39 The results obtained for the
C4-substituted furopyridine-3-ones (1–18) and related condensed
Model c-lactones (Fig. 1C) including 5,5-dimethyl-4-phenyl-5H-
furan-2-ones 19–2035,36 and 4-vinyllactones 212, 22,37 with various
functional groups at C3 and C4 were prepared as already described.
The condensed pyridine-dione d-lactone 23 (Fig. 1C) was synthe-
sized, starting from 4-methyl-2-oxo-2H-chromene-3-carbonitrile
and following steps i and iii as in Scheme 1A [condensation with
DMF DMA in xylene and further cyclization of obtained product
in AcOH/HCl (1:3) solution].38 The condensed pyridine-dione d-lac-
tone 24 was obtained from methyl 2,4-dimethyl-6-oxo-3,6-dihy-
dro-2H-pyran-3-carboxylate as described in Scheme 1C.
c-lactones (19–22) or pyridine-fused d-lactones (23, 24) are sum-
marized in Table 1. Compounds 1–18 show replacement of the
C4-carbonyl group normally present in cerpegin and furopyridine-
3,4-dione derivatives1,2 by a large diversity of chemical groups.
Among them are found: a hydroxyl group in nor-cerpegin (1,
Fig. 1B) and its analog 2, a sulfhydryl in 3, 4, a methyl in 5, 6, a
primary amine in 7–9, a secondary amine in 10, 11, 13–18 and a ter-
tiary amine in 12. Compounds 3, 5, 7 and 10–18 were dimethylated
at C1 as is the case for nor-cerpegin (1, this study) or for cerpegin.2 A
spiro-cyclohexane-fused group is the C1 substituent for compounds
Several new furo[2,3-d]pyrimidine-4-ones (25–31) (Fig. 1D) and
thieno[2,3-d]pyrimidine-4-ones 32–38 (Fig. 1E) were obtained via
dimethylaminomethyleneamino derivatives through pyrimidine
cycle formation. Furopyrimidinones were prepared as outlined in
Scheme 2A. Starting imino derivatives were synthesized by the
reaction of amides of cyanoacetic acid with the corresponding keto-
alcohol. Condensation of 2-imino-4,5,5-trisubstituted-2,5-dihydro-
furan-3-carboxamides XII with DMF DMA yielded the fused
compounds XIII. Thieno[2,3-d]pyrimidine-4-ones XV (35–44) were
synthesized in xylene from Gewald thiophenes (Scheme 2B and
Supplemental data).
2, 4, 6 and 9. Compound 10, bearing a benzylamino group at C4,
cPA
50
strongly inhibited the PA activity of c20S (IC of 600 nM). In the
range of 100 to 2000 nM, a selective inhibition of the c20S vs i20S
was found (no inhibition detected for the i20S, Table 1). At the
whole cell level, compound 10 also inhibited PA activity of HeLa

1574
A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
N
N
N
N
H
R
Cl
OH
R2
R1
A
iv
v
O
O
O
IV 1, 2
O
O
O
VI 10-18
V
iii
vi
N
N
NH2
CN
O
CN
O
R2
R1
R2
R1
R2
R1
O
i
ii
O
O
O
I
III 7- 9
II
vii
N
S
SH
NH2
R2
R1
R2
R1
O
i'
O
O
O
VIII 3, 4
VII
R1, R2 = CH3, CH3; CH3, C2H5; (CH2)5
N
N
B
C
O
O
R1
R2
R1
R2
R1
R2
i
ii
O
O
O
O
O
X
O
IX
XI 5, 6
R1, R2 = CH3, CH3; (CH2)5
OH
O
O
N
O
O
I
II
O
O
O
O
N
O
O
24
Scheme 1. Synthesis of 1,1-dialkyl-1H,5H-furo[3,4-c]pyridine-3-one 4-derivatives IV (1, 2), VIII (3, 4), III (7–9), VI (10–18) (A), XI (5, 6) (B) and related pyridinone-fused d-
lactone 24 (C). (A) Reagents and conditions: (i) I, dimethylformamide dimethylacetal (DMF DMA), xylene, reflux 5 h., 89–96% yield; (i0) VII, DMF DMA, heating 90 °C, 4 h,
yields 72–76%; (ii) II, ammonia 20%, reflux 4 h, yields 72–89%; (iii) II, AcOH/HCl (3:1), reflux 4 h, yields 94–99%; (i
ratio, xylene, reflux 2.5 h, b) V, NHR2, 1:33 molar ratio, DMF, reflux 2.5 h, yield 74%; c) V, NH2Ar 1:1 molar ratio, AcOH, reflux 10 h, yield 58–68%; (
phenoxymethyloxirane, 2–3 drops of water, isopropyl alcohol, reflux 20 h., yields 51–55%, ( ii) I, H2S, benzene, Et3N, 2 h. (B) Reagents and conditions: (i) IX, DMF DMA, xylene,
v
) IV (1), PCl5, 130 °C, 15–20 min; (
v
) a) V, NH2R 1:5 molar
v
i) III (7), 2-
v
reflux 3.5 h., 83–96% yield; (ii) X, ammonia 20%, reflux 5 h, yields 75–83%. (C) Reagents and conditions: (i) commercially available 2,4-dimethyl-6-oxo-3,6-dihydro-2H-pyran-
3-carboxylate, DMF DMA, xylene, reflux 5 h; (ii) obtained product, 20% ammonia, reflux 4 h.
O
O
O
3
3
R
NHR
3
NHR
O
A
B
R2
R1
N
R2
R1
+
NH
i
O
N
ii
O
CN
O
XII
XIII
25 - 34
O
O
O
R2
R2
R2
R3
N
O
3
NH2R
ii
O
R1
i
N
N
R1
N
35-44
S
S
XIV
NH2
R1
S
XV
Scheme 2. Synthesis of 1,1-dialkyl-5-methylene-5,6-dihydro-3H-furo[2,3-d]pyrimidine-4-ones XIII (A) and of 3-substitutedthieno[2,3-d]pyrimidine-4(3H)-one XV 35–44
(B) (A) Reagents and conditions: (i) amid of cyano-acetic acid, corresponding carbinol, ethyl alcohol, Sodium ethylate, room temperature, 24 h; (ii) XII, DMF DMA (1:1.2 molar
ratio), benzene/DMF, reflux 5 h, 52–78% yield. (B) Reagents and conditions: (i) ethyl 2-amino-thiophene-3-carboxylate, DMF DMA (1:1), xylene, reflux 7 h; (ii) ethyl 2-
(dimethylaminomethyleneamino)-thiophene-3-carboxylate XIV and NH2R 1:3 molar ratio, xylene, reflux 35 h.

A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
1575
Table 1
Inhibition of mammalian proteasomes by 4-R-1,1-disubstituted-1H,5H-furo[3,4-c]pyridine-3-ones (compounds 1–18, series III, IV, VI, VIII, XI) and by model compounds:
c
- (19–22) and pyridone-fused d-lactones (23, 24)
N
R
4
R1
R2
IC50 (lM) or % inhibition at 100 lM
Compound
1
O
O
R
Separate R1, R2 or spiro-fused R1/R2
CT-L
ni
T-L
PA
OH
1
2
3
4
5
6
ni
16.7 1.0
27.8 2.0
(48%)
OH
ni
ni
SH
SH
79
6
ni
(52%)
ni
ni
(20%)
88 12
ni
ni
ni
16.1 0.8
NH2
NH2
NH2
7
8
ni
(35%)
7,9 0,3
(21.5%)
ni
7.0 0.2
9
ni
ni
ni
ni
8.4 0.5
10
0.60 0.02 ⁄ni at 2
lM
NH
NH
S
11
(26%)
(29%)
(41%)
N
NH2
H
N
12
13
ni
ni
ni
(41.6%)
(41.5%)
H
N
Cl
(24.5%)
O
NH2
H
N
S
14
ni
ni
ni at 25 lM
O
OH
OH
H
N
15
16
ni
ni
48.1 0.7
ni
O
O
O
H
N
(36.5%)
60.8 9.6
OH
OH
H
N
18
18
(29%)
(63%)
ni
ni
ni
F
H
48.1 2.7
O
Cl
N
Other lactone-containing compounds
S
1935,36
ni
ni
ni
ni
ni
ni
NH2
O
O
2035,36
NH2
O
O
N
O
212
ni
ni
ni
O
O
O
(continued on next page)

1576
A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
Table 1 (continued)
N
R
4
R1
IC50 (lM) or % inhibition at 100 lM
Compound
1
O
R2
O
R
Separate R1, R2 or spiro-fused R1/R2
CT-L
ni
T-L
PA
ni
O
NH2
2237
2338
24
ni
O
O
NH
O
ni
ni
ni
ni
O
O
O
HN
29.4 1.8
8.7 0.8
O
O
Cerpegin2
ni
ni
0.0026
ni
ni
0.021
10.4 0.5
6.1 0.3a
0.46
Cerpegin N5-derivative1
SalA24
Inhibition of constitutive proteasome (c20S) from rabbit erythrocytes is expressed as IC50 values or as % inhibition at 100 lM (italics between parentheses). Inhibition of
immunoproteasome (i20S) from human blood monocytes is given for compound 10 and labeled with an asterisk⁄. ni = no inhibition. Cerpegin2, a N5 derivative of cerpegin1
and Salinosporamide A (SalA)24 are given as reference inhibitors.
a
IC50 values for compound 1 in Ref. 1.
Table 2
Inhibition of mammalian proteasomes by furo[2,3-d]pyrimidines (25–34, series XIII) and thieno[2,3-d]pyrimidines (35–44), series XV) derivatives
Compound
Furo- and Thieno[2,3-d]pyrimidine-4-ones
IC50
(
lM) or % inhibition at 100
lM
CT-L
ni
T-L
PA
ni
O
NH
25
26
27
28
59
ni
ni
ni
5
O
N
O
O
NH
ni
ni
ni
ni
N
O
N
NH
ni
O
O
N
(30%)
O
N
Br
O
29
ni
(29%)
ni
N
O
O
O
O
O
O
N
O
OH
N
N
N
N
N
30
31
32
33
ni
ni
ni
N
O
N
N
N
O
ni
(56%)
(48%)
ni
47 1.6
N
N
O
ni
ni
ni
O
O
N
O
(33%)
OH
OH
N
O
34
ni
ni
ni
N

A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
1577
Table 2 (continued)
Compound
Furo- and Thieno[2,3-d]pyrimidine-4-ones
IC50
(
lM) or % inhibition at 100
lM
CT-L
ni
T-L
PA
ni
O
N
35
36
(42%)
56 1.3
ni
S
S
N
O
Cl
ni
ni
N
N
O
N
N
N
37
38
39
ni
ni
ni
ni
ni
ni
S
S
S
N
O
N
42% at 50 lM
N
N
O
(53%)
N
O
N
Cl
O
9.9 0.4
⁄6.7 0.6
40
41
ni
ni
N
N
S
N
O
N
36% at 50
lM
19.4 1.9
25% at 50 lM
OH
S
N
Cl
Cl
Cl
O
42
43
44
ni
ni
ni
(48%)
ni
ni
ni
N
N
N
S
S
S
N
O
Cl
F
(52%)
N
O
56% at 50 lM
N
Inhibition of constitutive 20S proteasome from rabbit erythrocytes is expressed as IC50 values or as % inhibition at 100 lM (italics between parentheses). For some
compounds, % inhibition was tested at 50
l
M. Inhibition of immunoproteasome (i20S) from human blood monocytes is given for compound 40 and labeled with an asterisk⁄.
ni = no inhibition.
PA
50
cT-L
50
cervical cancer cell line (IC cel = 11.6 0.6
l
M). At these concen-
inhibition of T-L activity could be noted for 16 and 18 (IC
of
trations no cytotoxicity was found. Only higher concentrations
were found to be mildly cytotoxic (35% inhibition of mitochondrial
61
lM and 48
lM, respectively).
Remarkably, in this C4-substituted series of compounds derived
dehydrogenase activity at 50
l
M) and to decrease cell attachment.
from nor-cerpegin (1–18), an optimization of the inhibition po-
tency of PA activity was found for compound 10. Moreover, neither
this C4 benzylamino substituent in compound 10 nor other C4 sub-
stituents allow inhibition of the two other proteolytic sites with
the exception of 16 and 18 for which the T-L activity was slightly
inhibited. This was consistent with the previously reported ab-
sence of inhibition caused by the analogous N5-substituted com-
pounds derived from cerpegin1 which selectively inhibited the PA
activity of c20S.
Further analysis is necessary to determine this negative cellular ef-
fect of compound 10 more precisely.
Compound 14, with a 4-benzenesulfonamide substituent at C4
did not inhibit PA activity at high concentration (25 lM), in con-
trast with the N5-substituted analogous compound (Table 1, refer-
ence compound 11) which exerted submicromolar inhibition (ICc5P0A
of 6 lM). In the same manner compound 15, with a 4-(2-hydroxy-
3-phenoxy propylamino) group at C4, exerted very low inhibition
cPA
50
of PA (IC of 48
l
M). Similar results were obtained with the anal-
In the series of model compounds with related c-lactones (19–
ogous substitution at N5.1 When the above-mentioned phenoxy
propylamino group was declined with diverse substitutions in
para-position of the phenyl ring (-methyl in 16, -fluoro in 17
or -chloro in 18) PA inhibition was no longer detected. A slight
22) or pyridinone-fused d-lactones (23–24) (Fig. 1C and Table 1),
no inhibition of the three proteolytic activities was found for com-
pounds 19–22, ruling out the possibility that the pyridine ring of
the fused furo-pyridine-3-one structure could participate in PA

1578
A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
inhibition. Compound 24 behaved differently in that it inhibited
here for cPA is not very different from that reported for SalA on
the same activity (Table 124). In the series of furo- and thienopyr-
imidine-4-ones, compound 40 specifically inhibited T-L activity of
c20S or i20S, although at micromolar concentrations.
cT-L
CT-L and T-L mildly but not PA activity (IC
of 29 lM and
50
9
lM, respectively).
None of the compounds studied in this series (1–24) worked on
fractions of cathepsin B or calpain I.
Results for the assays with furo- or thienopyrimidine-4-ones
are summarized in Table 2.
All the compounds studied with a fused furopyrimidine ring
bear a carbonyle at C4 of the fused ring (25–34, Fig. 1D, Table 2).
With the exception of 25 and 31, which exerted mild inhibition
of T-L and PA activities, respectively, none of these compounds
were inhibitors of c20S. Compounds with a fused thienopyrimidine
ring (35–44, Fig. 1E, Table 2) also bear a carbonyl group at C4 of the
fused ring. In this series, no inhibition of the PA or CT-L activities
The binding mechanisms of compound 10 to the PA active sites
were studied performing in silico docking experiments (Fig. 2).
Catalytic b1 chains used as targets were from the constitutive
mouse 20S proteasome7 (Mmb1c, PDB ID: 3UNE, Fig. 2A) and from
the mouse immunoproteasome7 (Mmb1i, PDB ID: 3UNH Fig. 2B).
For comparison, we also included results obtained with b1 chains
from constitutive bovine 20S proteasome40 (Btb1c, PDB ID: 1IRU,
Fig. 2C) that have been used as target in our previous work for
analogous furopyridones. All crystal structures were obtained from
the protein data bank.41 These mammalian models were retained
due to the very strong amino acid sequence identity between hu-
man, mouse and bovine b-type subunits.42 For example, the bovine
b1 precursor protein is 95% identical to the human one and the
identity reaches 98.5% when considering only the mature subunit.
In addition, the few non-identical residues are located at the inter-
faces of b1 with its neighbors. The specificities of mouse immuno-
proteasome amino acid sequence have been described and
crystallographic data are available for mouse.7
was observed. However, mild inhibition of the T-L activity was
noted for compounds 36, 40 and 41 (IC
cT-L
50
of 56 lM, 10 lM and
19
l
M, respectively). The T-L inhibition of i20S by 40 confirmed
iT-L
50
the efficiency of this compound (IC
of 7 lM).
To summarize, in the series of furopyridine-3-ones, only the c-
lactone compounds fused to a pyridine ring substituted at C4 were
found to inhibit PA activity specifically in the c20S. The SH group at
C4 was unefficient as compared to the OH and NH2 groups at the
same position. Compound 10 with a C4-substituted benzylamino
group was the most potent inhibitor, working specifically on the
constitutive PA activity at the nanomolar range. For this activity,
The AutoDock Vina program43 was used for docking calcula-
tions,44 with default parameters. In cases where asymmetric car-
bons were present, all corresponding configurations were docked.
Visual Molecular Dynamics (VMD)45 was used to prepare
molecular pictures.
cerpegin c
-lactone2 and such an N5 derivative as compound 1 in
Ref. 1 (Table 1) were seventeen and ten times less efficient, respec-
tively. Although SalA b-lactone is a better inhibitor of CT-L and T-L
activities24, it is worth noting that the IC50 value of 10 observed
The lowest energy (first rank) poses adopted by the C4-substi-
tuted furopyridine 10 in the b1 catalytic sites of constitutive 20S
proteasomes (Mmb1c, Fig. 2A and Btb1c, Fig. 2C) were similar to
those already described for N5- and C1-substituted furopyridinones
in the bovine Btb1c site.1 The bicyclic furopyridine ring fitted into a
slit in the active site between Thr residues 1 and 21 (Fig. 2A and C),
and the C3 carbonyl group of the furan ring was positioned at a dis-
B
A
tance (4.2 Å) compatible with a nucleophilic attack by the O
c of
Thr1 (red dashed arrow in Fig. 2D). As previously observed for
the N5 substituent,2 the free end of the C4 substituent was close
to the aromatic ring of Tyr114 of the b2c subunit. On the contrary,
when 10 was docked in the mouse Mmb1i chain (Fig. 2B), the first
rank pose was different: the 4-benzylamino substituent was seen
to enter the S1 specificity pocket and the c-lactone ring was largely
Mm
displaced, pointing towards His114 of b2i. In this case, no relevant
interaction was observed between the inhibitor and the Thr1
nucleophile. This correlates with the fact that no inhibition was ex-
erted by compound 10 on the PA active site of the immune human
proteasome (Table 1). These observations strengthen the idea al-
ready expressed1,2 that Tyr114 in b2c could play an important part
in the specificity of binding to the PA active site.
Mm
O
+
H3N
4,2Å
D
N
H
C
T1
O
4,2Å
O
3,2Å
HN
O
N
OH
3,3Å
In this series of C4-substituted furopyridines, an 80-fold less
efficient compound (15) was also docked in Mmb1c and Btb1c sites
for comparison (Figs. S1 A and C) as well as in Mmb1i site (Fig. S1
B). For b1c sites, these assays revealed an orientation of the fused
furopyridine ring compatible with the same type of nucleophilic
attack as the one suggested for 10 in the same b1c sites, but the
fourth-rank pose did not correspond to the lowest energy one. Fur-
thermore, a different orientation was observed for the C4 substitu-
ent, the phenoxy ring of which deeply entered the S1 specificity
pocket. This highlights the importance of the ligand interaction
with Tyr114 in the b2c subunit for the inhibition efficiency on
the b1c catalytic site.
Whatever the enzymatic activity concerned, the necessary pre-
requisite for inhibition is ligand binding. In terms of binding, our
molecules may be compared with HU derivatives described by
Gallastegui et al.29 which non-covalently inhibit CT-L activity in
the yeast proteasome. The binding mode predicted for our mole-
cules converges with a novel one described by crystallography
NH
O
T21
T22
A49
Bt
Bt
A27
Y114
β2
Figure 2. In silico docking of compound 10 in the PA active site of mouse (A, B) and
bovine (C, D) 20S proteasomes. (A) Mouse constitutive PA site (Mmb1c and
neighboring Mmb2c subunits) (B) Mouse immunoproteasome PA site (Mmb1i and
neighboring Mmb2i subunits) (C) Bovine constitutive PA site (Btb1c and neighbor-
ing Btb2c subunits). Protein chains in A, B and C are shown as solvent accessible
surfaces and the residues and atoms relevant to an enzyme activity are labelled and
colored according to CPK convention. Compound 10 is displayed as stick model
colored by atom type. Each docking mode is shown as 6 superimposed poses from
the most populated and lowest energy cluster. (D) Distance map corresponding to
Figure 2C. Distances between the bovine b1c subunit and atoms in compound 10
were taken from the lowest energy pose. Putative hydrogen bonds are shown as
blue dashed lines and the predicted nucleophilic attack on the lactone carbon, by a
dashed red arrow.

A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
1579
for HU derivatives. All these molecules are rather small, non-pep-
tidic and share a clear separation of hydrophobic and hydrophilic
moieties. Their hydrophilic heads are pinched between Thr21
and Gly47 and form hydrogen bonds with the main chain atoms
of these residues at the entrance of the S1 subsite. Their hydropho-
bic tails engage in the S3 subsite, contributed in part by the neigh-
boring subunits (b2c for PA sites and b6 for CT-L sites). In addition,
the specificity of HU derivatives towards b5 is due to the presence
in these derivatives of a short hydrophobic branch that interacts
with residues in the S1 specificity pocket. The non-covalent nature
of the catalysis mechanism provided by HU derivatives is corre-
lated with their lack of interaction with the catalytic residue
Thr1,29 whereas docking predicts such interactions for compound
10 of this study (Fig. 2D).
the pose was very bad (8–10), in relation with the poor efficiency
of the compound in T-L inhibition (IC50 of 48 M).
l
Promising inhibitions of both constitutive and immuno T-L
activities were obtained after incubation with the thienopyrimi-
T-L
50
dine-4-one 40 (IC
9.9 lM and 6.7 lM, respectively, Table 2).
The catalytic b2 chains from the mouse 20S proteasomes (Mmb2c,
Fig. 3C and Mmb2i, Fig. 3D) were then used as targets for docking
experiments. Accordingly, docking revealed almost identical first
rank poses of 40 in the two active sites with the para-chlorophen-
ethyl substituent engaged in the S1 specificity pocket (Fig. 3C and
D). Beyond this, we found no pose suggesting the possibility of a
nucleophilic attack of this molecule by Thr1.
In conclusion, our results highlight the functional importance of
a c-lactone fused to pyridine ring in the selectivity of PA inhibition.
In silico docking suggests that the absence of inhibition of the
b1i activity by 10 is accompanied with an inversion of this binding
mode relative to b1c (Fig. 2A and C), with the hydrophilic head fac-
ing the S3 subsite and the hydrophobic tail entering the slightly
more hydrophobic S1 subsite (Fig. 2B).
The absence of inhibition of T-L activity by 10 is to be related to
its mode of binding to the b2 constitutive active site. Indeed, the
first rank pose obtained in the b2c active site of the bovine protea-
some (Fig. 3A) was almost identical to the one observed in the
mouse b1i site with the 4-benzylamino substituent deeply engaged
into the S1 specificity subsite and the furopyridine ring pointing
toward Asp125, which occupies in b3 the same spatial position
as His114 in b2i. This comparison gives support to the selective
inhibition of constitutive PA activity by 10. More canonical binding
modes of compound 18 to the b2c catalytic site are illustrated in
Fig. 3B. The position of the molecule remained compatible with
Indeed, compound 10 from the furopyridine-3-one family specifi-
cally targeted the constitutive PA sites. Comparison of this result
with the results previously reported with furopyridine-3,4-
diones1,2 show an optimization of 20S proteasome inhibition when
the C4 carbonyle is replaced by a benzylamino group. In spite of
this optimization, docking analysis suggests a unique mode of
binding with the C3 carbonyle of the furan ring positioned at a dis-
tance compatible with a nucleophilic attack by the Oc of Thr1. This
further suggests that the optimization noted above is not related to
different positions of the molecules in the active site but, rather, to
the details of intermolecular interactions. In contrast, in the thie-
no[2,3-d] pyrimidine-4-one family of compounds, that is, in the ab-
sence of the typical furopyridine-3-one ring, PA activities were not
inhibited. Moreover, in compound 40 of the same family, T-L activ-
ities were specifically reduced at the micromolar range in both the
constitutive proteasome and its immunoisoform.
the nucleophilic attack of the
c-lactone by Thr1 but the rank of
Acknowledgments
A.H., A.P., G.M. received financial supports from the Yerevan
University (A.H., A.P., G.M.) and from a Grant of the State Commit-
tee Science MES RA, in frame of the research project No. SCS 13-
1D330 (A.H., G.M.). T.H.P. received financial supports from the
Vietnamian Ministery for Education (T.H.P.). M.B.D., M.R.R., D.B.,
L.D., L.Q. and Y.C. received supports from the Pierre et Marie Curie
University (UPMC, Paris 6) and from a Grant of the ‘French Associ-
ation against myopathies’.
B
A
Supplementary data
Bt
Bt
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.
072.
C
D
References and notes
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Figure 3. In silico docking of compounds 10 (A), 18 (B) and 40 (C, D) in the T-L
active site of diverse 20S proteasomes. (A) Compound 10 in the bovine constitutive
T-L site (Btb2c and neighboring Btb3 subunits). (B) Compound 18 in the bovine
constitutive T-L site (Btb2c and neighboring Btb3 subunits). (C) Compound 40 in the
mouse constitutive T-L site (Mmb2c and neighboring Mmb3 subunits). (D)
Compound 40 in the mouse T-L site (Mmb2c and neighboring Mmb3 subunits).
Protein chains are shown as solvent accessible surfaces and the residues and atoms
relevant to an enzyme activity are labelled and colored according to CPK
convention. Compounds are displayed as stick model colored by atom type. Each
docking mode is shown as 3 (A) or 6 (B) superimposed poses (see text for details).
For compound 18, the reported configuration of the hydroxyl is S.

1580
A. Hovhannisyan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1571–1580
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c20S fractions were used. In summary, after a pre-incubation period of 15 min
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compounds (0.1–100
substrates ([S0] = 50
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l
M), reactions started after addition of the respective
l
M for CT-L and ([S0] = 100 M for PA and T-L activities)
l
l
M
20. Myung, J.; Kim, K. B.; Lindsten, K.; Dantuma, N. P.; Crews, C. M. Mol. Cell 2001, 7,
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16, 1278.
Tris, 1 mM DTT, 10% glycerol for T-L activity. In vitro measurements of iPA and
iT-L activities were realized using i20S fractions from human erythrocytes and
the buffers described above for the homologous PA and T-L activities. A 2-min
pre-incubation period was used for each activity and initial concentrations for
the respective Z-LLE-bNA and Boc-LRR-AMC substrates were as follows:
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I.; van der Marel, G. A.; Kaiser, M.; Kisselev, A. F.; Overkleeft, H. S. Org. Biomol.
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[S0] = 50
and cathepsin
respective Suc-LLVY-AMC and Z-RR-AMC substrates. Buffers and temperatures
used were: 50 mM Tris, 100 M CaCl2 and 10 mM DTT (pH 7.2) at 25 °C for
calpain I, and 352 mM KH2PO4, 48 mM Na2HPO4, 1 mM EDTA, 1 mM DTT (pH
6) at 37 °C for cathepsin B. In all enzymatic assays, emitted fluorescence was
measured using black 96-well microplates and a BMG Fluostar microplate
reader. The inhibitor concentrations giving 50% inhibition (IC50 values) were
l
M and 100
lM, respectively. In vitro measurements of calpain I
B
activities were realized using human fractions and the
l
obtained by plotting the percent inhibition against inhibitor concentration to
nH
equation:
%
inhibition = 100[I]/(IC + [I]nH),
or
equation:
%
50
inhibition = 100[I]nH/((IC5n0H + [I]nH) where nH is the Hill number.; (b) In vivo
measurements of the PA activity in HeLa cervical cancer cell line were
conducted with the proteasome cell-based assay from Promega. HeLa cells
were incubated in DMEM medium supplemented with the inhibitor for 17 h at
37 °C. White 96-well microplates were seeded with 5000 cells in 100 lL DMEM
per well. Control assays were made in the same conditions, without inhibitor. A
second step of incubation was realized at 20 °C for 1 h in order to place cells at
the optimum temperature for the reactions to reveal proteasomal activity.
Measurements of intracellular PA activity were done by adding a 1:1 (vol/vol)
mixture of the PA-substrate Z-nLPnLD conjugated to aminoluciferine and
luciferase to the cells. Chemiluminesence measurements were done after 5-
min reaction using a BMG Fluostar microplate reader. In-cell IC50 values (IC50
cel) were calculated as described above.; (c) Cytotoxicity assays were
conducted using the reduction capacities of mitochondrial dehydrogenases
from living cells to turn a colorless tetrazolium salt solution (XTT from Sigma)
into an orange solution of formazan. HeLa cells were prepared as described for
measurements of in-cell proteasomal activities and incubation periods with
inhibitor were of 18 or 72 h. Readings were made by spectrophotometry at
485 nm after a 3-h period of reaction with a 1 mg/mL XTT solution.
40. Unno, M.; Mizushima, T.; Morimoto, Y.; Tomisugi, Y.; Tanaka, K.; Yasuoka, N.;
Tsukihara, T. Structure 2002, 10, 609.
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Groll, M.; Vidal, J.; Reboud-Ravaux, M. J. Med. Chem. 2013, 56, 3367.
35. Communication by Melikyan, G., Hovhannisyan, A. and Avetisyan, A. at the
Congress of the French Chemical Society «La Chimie du Futur. Le Futur de la
Chimie» SFC 07, Paris, July 16–18 2007.
41. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
36. (a) Avetisian, A.A.; Melikian, G.S., Danghian, M.T., The inventor’s certificate,
USSR, N 424425 (11.11.1971).; (b) Knott, E. B. J. Chem. Soc. 1948, 186.
37. Perjessy, A.; Avetisian, A. A.; Akhnazarian, A. A.; Melikian, G. S. Chem. Commun.
1989, 54, 1666.
38. Mandal, T. K.; Kuztenov, V. V.; Soldatenkov, A. T. Chem. Heterocycl. Compd.
1994, 30, 867.
42. UniProt Consortium Nucleic Acids Res., 2012, 40, D71.
43. Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455.
44. The docking space was limited to a 20 ꢀ 20 ꢀ 20 Å box centered on the N
terminal threonine of the catalytic subunits. Docking modes were selected on
the basis of the distance (3–4 Å) between the carbonyl carbon of the compound
lactone and the hydroxyl oxygen of the N terminal threonine of each subunit.
45. Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33.
39. (a) In vitro measurements of CT-L, T-L and PA activities of the constitutive
proteasome were realized as previously described in Ref. 1,2 mainly using c20S