Note: Descriptions are shown in the official language in which they were submitted.
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Proteasome inhibitors
The present invention relates to a compound of formula (I)
R2
Ri X 1( Z
R3
(I),
wherein
X is C=0, C=S or B-OH;
Y is an electrophile and Z is a leaving group, or Y¨Z is an electrophile;
R1 comprises or consists of
(a) (i) a first group binding to a proteolytic site of a proteasome, said
first group being
bound to X; and
(ii) optionally a second group enhancing delivery;
or
(b) a group binding between subunits (31 and p2 of a proteasome;
R2 and R3 are independently selected from H, methyl, methoxy, ethyl, ethenyl,
ethinyl and
cyano, wherein methyl and ethyl may be substituted with OH or halogen.
In this specification, a number of documents including patent applications and
manufacturer's
manuals are cited. The disclosure of these documents, while not considered
relevant for the
patentability of this invention, is herewith incorporated by reference in its
entirety. More
specifically, all referenced documents are incorporated by reference to the
same extent as if
each individual document was specifically and individually indicated to be
incorporated by
reference.
In recent years, the proteasome has been validated as a therapeutic target for
anti-cancer
therapy and the first proteasome inhibitor Bortezomib has been approved in
2008 for the
treatment of multiple myeloma and mantle cell lymphoma. Current efforts focus
on the
development of second generation inhibitors with improved pharmacological
properties.
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Epoxyketone inhibitors of the proteasome are desirable cancer therapeutics.
The dose
required to elicit inhibition is considerably lower than the boronic acid
proteasome inhibitor
Bortezomib. Additionally, many patients treated with Bortezomib acquire
resistance to this
agent through mutations in the 13-subunits, i.e. the active site subunits of
the proteasome. It
has been recognized in recent years that Bortezomib resistant patients are
responsive to
Carfilzomib, which is the only epoxyketone inhibitor approved for clinical use
today. This
further highlights the importance of the development of additional epoxyketone
inhibitors
and/or inhibitors exhibiting similar inhibition properties as epoxyketone
inhibitors. In pursuit of
this goal most approaches today focus on the modification of the peptide
backbone of
epoxyketone inhibitors. These modifications include the introduction of
natural and non-
natural amino acids to increase the specificity for a given proteasome active
site, the
solubility of epoxyketone inhibitors, the bioavailability and the possibility
to orally administer
cancer therapeutics. In addition, medicinal chemistry approaches have focused
on the
addition of capping agents at the S4 position, which increase the absorptive
properties of
epoxyketone inhibitors and/or protect them from the harshly acidic environment
of the gastro-
intestinal tract.
The parent molecule of this class epoxyketone proteasome inhibitors,
epoxomicin, was first
isolated as a natural product synthesized by an Actinomyces bacterial strain.
Later, another
molecule of this class was found, viz. eponemycin, and several synthetic
variants of these
molecules (Figure 1) are presently in clinical trials as cancer therapeutics
(Bennett and Kirk,
Curr. Opin. Drug Discov. Devel., 11, 616-625 (2008); Demo et al., Cancer Res.,
67, 6383-
6391 (2007)).
The efficacy of epoxyketone inhibitors, is a consequence of the dual
electrophile nature of
the epoxyketone group. Based on several co-crystal structures of epoxyketone
inhibitors with
yeast, mouse and human 20S proteasomes, it has been described that the y-
hydroxyl group
of the proteasome active site catalytic threonine reacts with the ketone
moiety, whereas the
N-terminal amino group of Thrl reacts with the carbon atom of the epoxide of
the inhibitor,
which is in a-position to the ketone, in an irreversible manner. As shown in
Scheme 1, this
suggested chemistry results in the formation of an 1,4-morpholine ring
product, which is
formed by the active site catalytic threonine and the epoxyketone inhibitor
(Groll et al.,
Journal of the American Chemical Society, 122, 1237-1238 (2000)).
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Lyses3,
= NH2
0,
1-1
HO 4,0
=a, HO
0
Thr Thr2 14,
1 oil,' "== 5 3
40. N %2
=== -6412 4
Thr2
0 H2 /10 0
OH 0
CAT
Scheme 1: Proposed chemical reaction mechanism for the inhibition of
proteasome active
sites by epoxy ketone inhibitors (Groll et al., /oc. cit.) Initially, the y-
hydroxyl group of the
actives site catalytic threonine amino acid side chain esterifies the ketone
group of the
epoxyketone inhibitor by a nucleophilic attack (left). The nucleophilic
addition reaction results
in the formation of a hemiketal intermediate where the y-hydroxyl group of the
active site
catalytic threonine is reversibly bound to the epoxyketone inhibitor (middle).
The N-terminal
amino group of the active site catalytic threonine then reacts with the
electrophilic carbon
atom in a-position to the ketone to form the covalently modified proteasome
active site
(right). This 1,4-morpholine ring structure which is formed by both the
proteasome active site
and the inhibitor results in an irreversible inhibition of the proteasome
active site.
This proposed inhibition scheme was formulated based on crystal structures
where the
electron density in the 20S proteasome active site was of insufficient
resolution and quality to
unequivocally model the inhibited state in atomic detail. Yet, the common
belief that
irreversible proteasome inhibition by epoxyketones yields a six atom ring
adduct is well-
established in the art; see, for example, the section dedicated to Carfilzomib
and references
cited therein in the review about proteasome inhibitors by Kubiczkova et al.
(J. Cell. Mol.
Med., 18, 947-961 (2014)).
There remains a significant need to develop new proteasome inhibitors, in
particular
improved proteasome inhibitors, improvements including activity at lower
dosage and less
side effects.
This technical problem has been solved by the subject-matter of the enclosed
claims.
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Accordingly, the present invention, in a first aspect relates to a compound of
formula (I)
R2
Y.
Ri X Z
R3
(I),
wherein
X is C=0, C=S or B-OH;
Y is an electrophile and Z is a leaving group, or Y¨Z is an electrophile;
R1 comprises or consists of
(a) (i) a first group binding to a proteolytic site of a proteasome, said
first group being
bound to X; and
(ii) optionally a second group enhancing delivery;
or
(b) a group binding between subunits 131 and 132 of a proteasome;
R2 and R3 are independently selected from H, methyl, methoxy, ethyl, ethenyl,
ethinyl and
cyano, wherein methyl and ethyl may be substituted with OH or halogen.
It is understood that throughout the present specification the term "compound"
encompasses
pharmaceutically acceptable salts, solvates, polymorphs, prodrugs, codrugs,
cocrystals,
tautomers, racemates, enantiomers, or diastereomers or mixtures thereof unless
mentioned
otherwise.
The compounds of formula (I) have a bipartite structure. R1, herein also
referred to as
"targeting group", is responsible for targeting the compound of formula (I) to
the proteolytic
site of the proteasome. This part of the compound may build on established
knowledge; for
details see below. The remainder of said compound is displayed in more detail
in formula (I).
This part is also referred to as "headgroup" or "warhead" herein. It is
responsible for the
irreversible inhibition of the proteasome. The headgroup requires an
electrophile (Y or Y¨Z)
in 13-position relative to the functional group X. Such a design is contrary
to the common
belief that irreversible inhibition of the proteolytic site of the proteasome
yields a morpholine
ring. To explain further, as can be seen in Scheme 1 displayed herein above,
the expected
mechanism involves a nucleophilic attack of the amino group of Thr1 on a
position which is in
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PCT/EP2017/063699
,
a to the ketone of the epoxyketone inhibitor. The epoxy group is amenable to
such
,
nucleophilic attack, noting that the carbon in a-position to the ketone is
electrophile.
,
,
'
The present inventors determined more than 20 crystal structures of the human
20S
,
,
proteasome at improved resolutions ranging from 1.8 ¨ 2.2 A. These new crystal
structures ,
of human 20S proteasomes provide insight into the inhibited state at atomic
resolution. In '
contrast to the previously described formation of a 6-membered 1,4-morpholine
ring structure ,
for epoxyketone proteasome inhibitors, it is now possible to visualize the
formation of a 7-
membered 1,4-oxazepane ring structure involving the catalytic threonine
residue of the
,
proteasome active site and the inhibitor in the inhibited state. Formation of
such an 1,4-
oxazepane inhibited state is achieved by a double electrophilic reactive group
on the
inhibitor, where both electrophiles are in a distance of two atoms to one and
another.
,
This finding allows to propose a novel chemical mechanism for the inhibition
of 20S
proteasomes by epoxyketone inhibitors. In this reaction mechanism, initially
the y-hydroxyl
,
,
group of the proteasome active site catalytic threonine reacts with the ketone
moiety, similar
to the situation where the 1,4-morpholine ring (6-ring) structure is formed.
In contrast to
established theory, and to allow for 1,4-oxazepane 7-ring formation, the N-
terminal amino
group of Thr1 reacts in an irreversible manner with that carbon atom of the
epoxide of the
inhibitor which is in 3-position to the ketone (Scheme 2). The reaction with
the carbon atom
of the epoxide in 3-position to the ketone appears to be favored because it is
the less
,
substituted center.
,
,
,
Lys33,1
[9
NUK .. = NH2
0 e
HO %0
1.06 HO
1 R
0 414... --N.
..., - .
-I-
01 N
11....c
H2 Thr2 I
% 0 .. .... A
HiN el iii 0 Thr2
R Alµ%4)1414 Thr2
HO i 4 3
i N
H 0
CAT
Scheme 2: Chemical reaction mechanism for the inhibition of proteasome active
sites by
epoxyketone inhibitors according to the invention and based on high resolution
co-crystal
,
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PCT/EP2017/063699
structures with human 20S proteasomes. Initially, the y-hydroxyl group of the
active site
catalytic threonine amino acid side chain esterifies the ketone group of the
epoxyketone
inhibitor by a nucleophilic attack (left). The nucleophilic attack results in
the formation of a
hemiketal intermediate, where the y-hydroxyl group of the active site
catalytic threonine is
reversibly bound to the epoxyketone inhibitor (middle). Until this step, the
reaction
mechanism for the formerly assumed 1,4-morpholine and the newly evidenced 1,4-
oxazepane structures in the inhibited state are identical. For the formation
of the 1,4-
oxazepane structure, however, the N-terminal amino group of the active site
catalytic
threonine then reacts with the electrophilic carbon atom of the epoxide in 13-
position to the
ketone to form the covalently modified proteasome active site (right). This
1,4-oxazepane
ring structure, which is formed by both the proteasome active site and the
inhibitor results in
an irreversible inhibition of the proteasome active site.
From a theoretical perspective, epoxyketone inhibitors are capable of forming
either type of
adduct, namely a 6-ring or a 7-ring. However, the formation of a 7-ring adduct
has never
been considered. Instead, the prior art is consistent in that a morpholine
ring would be
formed. The present inventors' work is first to establish that a 7-ring adduct
is formed. As a
consequence, the present inventors are the first to disclose the specific
molecular
architecture as laid down in formula (I) which is specifically tailored for
the formation of a 7-
ring adduct. The inhibitory activity of the art-established epoxyketones
entirely fails to
suggest this mechanism. Only in view of the high resolution crystal
structures, the present
inventors were able to discover the 6-regiospecificity of the nucleophilic
attack of the active
site threonine residue of the proteasome.
Without wishing to be bound to a specific theory, the present inventors
furthermore consider
that the formation of a seven ring adduct is kinetically favoured. Therefore,
the provision of
the compounds of the first aspect of the present invention which are
specifically tailored for
said kinetically favoured reaction mechanism will allow the use of lower
dosages. As a
consequence, fewer side effects are expected.
X is a functional group providing an electrophile. It can be a keto group, a
thio-keto group or
a boronic acid group. Deviant from the boronic acid group as present, for
example, in
Bortezomib, the compound of formula (I) requires that boron is part of the
main chain. As
shown in Scheme 2, the oxygen of the hydroxy group of the active site
threonine residue of
the proteasome binds to the electrophile atom in group X (the carbon atom or
the boron
atom).
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The group Y¨Z defines a second electrophile. Either Y as such or Y¨Z as a
whole is
electrophile; for preferred implementations see further below.
Moieties R2 and R3 define the intervening portion of the compound between the
two
electrophiles.
R1 is the above mentioned targeting moiety. Targeting moieties are known in
the art. In many
instances, targeting moieties are peptidic in nature. This applies, for
example, to Epoxomicin,
Oprozomib, Carfilzomib, Dihydroeponomycin, Eponemycin and ONX-0914. Since the
active
site of the proteasome exhibits proteolytic activity, peptides generally bind
to said active site
and are accordingly useful for targeting.
Said first group may optionally be bound to a second group which second group
enhances
delivery. To the extent the first group is peptidic in nature and consists of
three amino acids
or derivatives thereof, said second group may occupy a fourth position, in the
art also
referred to as S4 position. Dorsey et al. (J. Med. Chem., 51, 1068-1072
(2008)) and Zhou et
al. (J. Med. Chem., 52, 3028-3038 (2009)) explored several suitable groups
which are
attached to the N-terminal end of a peptidic first group. These groups
attached to the N-
terminal end of a peptidic first group as described in these two publications
all constitute
suitable second groups in accordance with the present invention.
An alternative term for said second group is "N-cap".
While peptidic first groups are envisaged, the particular structure of the
targeting group is not
key to the present invention. Basically, any chemical group which provides for
targeting to a
proteolytic site in the proteasome or to the vicinity thereof is a suitable
group R1 in
accordance with the present invention. To the extent a targeting group does
not target
directly to the proteolytic site, but to another site of the proteasome in the
vicinity of the
proteolytic site, linkers of suitable length may be used to connect the
targeting group R1 to
the active element of the compound of the first aspect, said active element
comprising
groups X, Y and Z.
To give an example of alternative targeting groups, Beck et al. (Angew. Chem.
Int. Ed. 54,
11275-11278 (2015)) describe a molecule binding to the crevice between 01 and
62 subunit
of the proteasome. This compound is displayed below.
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o K
0. N
c . 441ii
0
Replacing the ethyl group of the ethoxy moiety bound to the phenyl group of
said compound
is a means of rendering this molecule suitable group R1. This is explained in
further detail
below.
The term "pharmaceutically acceptable salt" refers to a salt of a compound of
the present
invention. Suitable pharmaceutically acceptable salts include acid addition
salts which may,
for example, be formed by mixing a solution of compounds of the present
invention with a
solution of a pharmaceutically acceptable acid such as hydrochloric acid,
sulfuric acid,
fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric
acid, tartaric acid,
carbonic acid or phosphoric acid. Furthermore, where the compound carries an
acidic
moiety, suitable pharmaceutically acceptable salts thereof may include alkali
metal salts
(e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium
or magnesium
salts); and salts formed with suitable organic ligands (e.g., ammonium,
quaternary
ammonium and amine cations formed using counteranions such as halide,
hydroxide,
carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate).
Illustrative
examples of pharmaceutically acceptable salts include, but are not limited to,
acetate,
adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bicarbonate, bisulfate,
bitartrate, borate, bromide, butyrate, calcium edetate, camphorate,
camphorsulfonate,
camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate,
digluconate,
dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate,
ethanesulfonate,
formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate,
glycerophosphate,
glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate,
hydrabamine,
hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate,
hydroxynaphthoate,
iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,
maleate, malonate,
mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-
naphthalenesulfonate,
napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate,
oxalate, pamoate
(embonate), palmitate, pantothenate, pectinate, persulfate, 3-
phenylpropionate,
phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,
salicylate, stearate,
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sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate,
triethiodide, undecanoate,
valerate, and the like (see, for example, S. M. Berge et al., "Pharmaceutical
Salts", J. Pharm,
Sc., 66, pp. 1-19 (1977)).
When the compounds of the present invention are provided in crystalline form,
the structure
can contain solvent molecules. The solvents are typically pharmaceutically
acceptable
solvents and include, among others, water (hydrates) or organic solvents.
Examples of
possible solvates include ethanolates and iso-propanolates.
The term "codrug" refers to two or more therapeutic compounds bonded via a
covalent
chemical bond. A detailed definition can be found, e.g., in N. Das et al.,
European Journal of
Pharmaceutical Sciences, 41, 2010, 571-588.
The term "cocrystal" refers to a multiple component crystal in which all
components are solid
under ambient conditions when in their pure form. These components co-exist as
a
stoichiometric or non-stoichometric ratio of a target molecule or ion (i.e.,
compound of the
present invention) and one or more neutral molecular cocrystal formers. A
detailed
discussion can be found, for example, in Ning Shan et al., Drug Discovery
Today, 13(9/10),
2008, 440-446 and in D. J. Good et al., Cryst. Growth Des., 9(5), 2009, 2252-
2264.
The compounds of the present invention can also be provided in the form of a
prodrug,
namely a compound which is metabolized in vivo to the active metabolite.
Suitable prodrugs
are, for instance, esters. Specific examples of suitable groups are given,
among others, in
US 2007/0072831 in paragraphs [0082] to [0118] under the headings prodrugs and
protecting groups.
To the extent compounds of the invention exhibit a pH-dependent charged state,
it is
understood that all possible charged states are embraced. A preferred pH range
in this
regard is from 0 to 14.
To the extent a compound according to the invention bears a net charge, it is
understood that
the compound is provided in electroneutral form. This is achieved by one or
more
counterions, preferred counterions being defined in relation to the term
"salt" herein above.
In a preferred embodiment, said first group is a peptidic group comprising or
consisting of at
least two amino acids, or a corresponding peptidomimetic, wherein X takes the
place of the
carbonyl group or of the a-carbon of the C-terminal amino acid of said
peptidic group, and
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said second group, if present, is bound to the N-terminus of said peptidic
group.
Preferred amino acids are a-amino acids. Preferred a-amino acids are the 20
proteinogenic
amino acids. Also preferred are derivatives of said proteinogenic amino acids,
for example,
the methyl ester of serine, also denoted "Ser(OMe)". Other derivatives of the
proteinogenic
amino acids may be used as well, for example those described in Zhou et al.
/oc. cit. and
including 4-diazolyl-alanine, homoserine methyl ester, pyridyl alanines such
as 2-pyridyl
alanine, 3-pyridyl alanine and 4-pyridyl alanine; 3-thienyl alanine,
cyclohexyl alanine, cyano
alanine and methyl serine.
Also other a-amino acids such as 2-amino butyric acid may be used.
In further preferred embodiments, 8-amino acids may be used in one or more
positions of the
peptidic group. Also preferred is to use D-amino acids at one or more
positions.
As an alternative to peptide bonds, alternative functional groups may be used.
This may
apply to a single peptide bond or to two or more peptide bonds. If all peptide
bonds are
replaced by other functional groups, the backbone chemistry in its entirety is
changed.
Alternative backbones are known in the art and include polyactide (PLA),
alkylamines,
jeffamines and those shown below ((a) to (r)) and described in Grimm et al.
(Acta Cryst D
(2010), 66, 685-697).
i
I
CA 03026564 2018-12-04
I
W02017/211818 11 PCT/EP2017/063699 1
I
i
i
2
I X 0
1
0 0 X k
1
1i
112 i
k
....,_ __ ......
\
0 ___________ 1
Otz,..' I
s'''..."...10
0
0 i
i
i
es
t 1
Sk
1
./
0
--:- .. p 0
,
\
f
t
i
,
0 .... -
X 6
,
1.: 1
;
,
X .
I - --- --1
i
X X
(-- __________________________________
0..
0 ... ): s 0
()
0 ' g..1
d
=====
.,
0
X frirOZ
Z
(.... I t
i
..... I. __
0
N
X
Z
.
1.---: F¨
.*: ¨1 i
;
0
(!) .
:
,:....
.....õ
-----"-------ik te
L........___ ..._____J .t
o
I
,
,
i
:
t
;
s
I
CA 03026564 2018-12-04
i
W02017/211818 12 PCT/EP2017/063699
1
i
;
i
Z
i
0
;
,
i
fil s.,
f
i
I
9--- =-.
k
,
o i
I
o
i
t
!
'
i
I
I
,=-=., ...,==='f'.2\.......H._______I
er, 0
,
t
1
:
t e;
.a.
....
Z W
4.
,
.
.
X
X X
.it)
:Ci .--.
Y ____________________________ 0
...
,
. :...7. x cr"
x
_
0 0
0
(II:3
27 Z 0
I I
,
CA 03026564 2018-12-04
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PCT/EP2017/063699
The above displayed backbones are as follows.
(a) M-type Jeffamines. R1 = ¨H for ethylene oxide (EO) or ¨CH3 for propylene
oxide (PO).
The P0/E0 molar ratio is 29/6 for Jeffamine M2005, 10/31 for Jeffamine M2070
and 9/1 for
Jeffamine M600. (b) Pentaerythritol ethoxylate. (c) Pentaerythritol
propoxylate. (d) Polyvinyl
pyrrolidone. (e) Polypropylene glycol. (f) Polyvinyl alcohol. (g)
Polyacrylate. (h) Cellulose
-
based polymers. R1 = ¨H, ¨CH3 or ¨CH2CHOHCH3 (hydroxypropyl methylcellulose),
¨H
or CH2CO2H (carboxymethyl cellulose). (i) Poly(ethylene imine). (j)
Di[poly(ethyleneglycol)]
adipate. (k) Jeffamine ED2003. (I) Jeffamine D2000. (m) Jeffamine SD2001. (n)
T-type
Jeffamines. (o) Polyacryl amide. (p) Glycerol ethoxylate. (q) Acrylic
acid/maleic acid
copolymer. (r) Vinylpyrrolidone/vinylimidazole copolymer. Each of the indices
x, y, z and w is
independently chosen from the integer numbers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and
10, provided
that a minimal length of at least two, preferably three building blocks is
obtained. While
branched backbones such as those depicted in (b) and (c) are not excluded,
preference is
.. given to backbones which are not branched. Accordingly, in the formulae for
branched
backbones such as (b) and (c), preference is given to two indices selected
from x, y and z
being 0. Potential positions for amino acid side chains are denoted as
ellipses in the figure.
These positions correspond to the Ca atmons in a peptide.
More generally speaking, the peptidic group may be replaced by a corresponding
peptidomimetic. The term "corresponding" in this context means that the
peptidomimetic has
similar size when compared to a peptidic group. Accordingly, a peptidic group
consisting of
two amino acids and a peptidic group consisting of three amino acids,
respectively, each
impose size limits on a corresponding peptidomimetic. A peptidomimetic in
accordance with
the present invention may use the above mentioned alternative backbones and/or
any of the
mentioned non-proteinogenic amino acids, noting that amino group and/or
carboxylic group
of any non-proteinogenic amino acid may be further modified in order to
account for the
mentioned alternative backbone.
In terms of secondary structure, preference is given to peptidic groups,
corresponding
peptidomimetics or backbones which are capable of assuming a 13-sheet
structure. Specific
backbones capable of assuming a I3-sheet structure include peptide bonds and
the
alternatives described above.
As disclosed above, X may take the place of the carbonyl group of the a carbon
of the C-
terminal amino acid of said peptidic group. In other words, and assuming that
X is CO which
is preferred, X is the carbonyl group of said C-terminal amino acid. In the
alternative, the
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PCT/EP2017/063699
a-carbon may be replaced with X. In that case, the C-terminal amino acid of
said peptidic
group may be viewed as a truncated amino acid. Having said that, it is
formally considered
as a separate building block and is also referred to as S1 site of the
proteasome inhibitor.
The term "peptidic" refers to peptide bonds connecting the amino acids of said
group. The C-
terminal end of the peptidic group is the end which is connected to X. To the
extent the
peptide bonds are inverted, i.e. NHCO instead of CONH, this is a preferred
embodiment
falling under the term "peptidomimetic".
Detailed exemplary synthesis procedures for two preferred compounds are given
in the
examples. In the following, general synthesis procedures are disclosed.
Synthesis of active ester: A) Beta-Keto acid formation from Boc-L-amino acid,
esterification
for the protection of beta-carboxyl moiety. B) Methylation reaction. C) Boc-
Deprotection. D)
Peptide coupling. E) Saponification of ester to Beta-Keto acid (partially
instable). F)
Preparation of an active ester with N-Hydroxysuccinirnide.
Synthesis of a Beta-Ketoaldehyde: G) Preparation of a Weinreb-Amide from Boc-L-
amino
acid. H) Gringard reaction to obtain alpha-Dioxacyclopentyl Ketone.
Methylation Reaction. I)
Boc-Deprotection. J) Peptide coupling. K) Oxidation to Beta-Ketoaldehyde.
Tripeptide Synthesis: L) Protection of the carboxyl group of Boc-L-Amino Acid
by
esterification. M) Boc-Deprotection. N) Peptide coupling with Boc-L-amino
acid. 0) Boc-
Deprotection. P) Coupling with N-cap acid. Q) Saponification of ester to acid.
As an alternative to R1 being a peptidic group or a peptidomimetic, a group
binding between
subunits 131 and 132 of the proteasome may be used. Preferably, said group is
an aryl
sulfonamide, aryl preferably being phenyl, aryl being connected to R12 as
defined in the
following. Between said aryl sulfonamide and R12 further moieties may be
present, said
further moieties preferably being isosteric with the corresponding moieties of
the particularly
preferred group binding between subunits 131 and 132 as disclosed in the
following. Particuarly
preferred is that said group binding between subunits 131 and 132 is 442-(4-
R12-oxy-phenyl)
quinolin-4-y1 carboxamido] benzene N-acetyl sulfonamide. R12 is a linker
connecting said
group binding between subunits 131 and 132 to X, preferably being C5 to C10
alkyl, C5 to C10
alkenyl or ¨[0-(CH2)2]1-5, wherein one or more C atoms in said linker may be
replaced with 0
and/or N.
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Said alkenyl group may comprise one, two or more double bonds.
So far, the group Y¨Z has been displayed as a double bond, wherein one of the
two bonds
comprised in the double bond is dashed. This indicates that Y and Z may either
be
connected by a double bond or a single bond.
In a preferred embodiment, Y¨Z is Y=Z, and preferably selected from CH=0 or
CH=CH2. In
an alternative, Y¨Z is Y¨Z and is preferably selected from CH2-1, CH2-Br, CH2-
CI, CH2-
OPO(OH)2, CH2-0Ts or CO-NHS wherein OTs is p-toluene sulfonyloxy and NHS is N-
oxy-
succinimide. As an alternative to the NHS-activated ester, also other
activated esters known
in the art may be used.
In a further alternative preferred embodiment, Y¨Z is Y¨Z and preferably
selected from 0-1,
0-Br, 0-CI, S-1, S-Br and S-I.
In a further preferred embodiment, R2 and R3 are identical and preferably
methyl, H, methoxy
or ¨CH2OH. Particularly preferred is that both R2 and R3 are methyl or that
both R2 and R3
are H. Especially preferred is that both R2 and R3 are methyl.
Preferred is that X is CO.
Preferred is that Y¨Z is CH=0. Also preferred is that Y¨Z is CO-NHS.
Particularly preferred is that X is C=0 and Y¨Z is CH=0 or CO-NHS.
Particularly preferred is that X is C=0 and Y¨Z is CH=0 or CO-NHS and both R2
and R3 are
methyl.
The following preferred embodiments are dedicated to preferred implementations
of the
targeting moiety R1. Any of the preferred embodiments of R1 can be combined
with any of
the preferred embodiments of X, Y, Z, R2 and R3.
In a preferred embodiment, said peptidic group consists of three a-amino
acids, wherein
preferably (a) the N-terminal amino acid is selected from Ser(OMe), Leu, Phe
and Ala; the
middle amino acid is selected from Ser(OMe), Leu, Phe and Ala; and/or the C-
terminal amino
acid is selected from Phe, Tyr, Leu, Ser(OMe) and Ala; or (b) said peptidic
group consists of
Ser(OMe)-Ser(OMe)-Phe, Leu-Leu-Tyr or Ala-Ala-Ala.
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Said N-terminal amino acid is also referred to as the moiety defining the S3
site of the
inhibitor. Said middle amino acid defines the S2 site. Said C-terminal amino
acid defines the
S1 site.
In a further preferred embodiment, said group enhancing delivery is present
and is
R11-00, !I .-01 -
CS-, or R11-S02-,
R11 being selected from the following substituted or unsubstituted groups,
groups being
carbocyclyl, heterocarbocyclyl, carbocyclyl alkyl, heterocarbocyclyl alkyl,
alkyl
heterocarbocyclyl alkoxy heterocarbocyclyl, alkoxyalkyl heterocarbocyclyl,
heterocarbocyclyl
amino, heterocarbocyclyl alkyl heterocarbocyclyl, and alkyl heterocarbocyclyl
alkyl
heterocarbocyclyl,
wherein alkyl is C1 to C4 alkyl, preferably methyl, substituents are C1 to C4
alkyl, preferably
methyl or ethyl, C1 to C4 alkoxy, preferably methoxy or ethoxy, hydroxy and/or
halogen,
preferably Cl, Br or I, and heteroatoms are 0, N and/or S.
R11-CO is particularly preferred.
R11 being heterocarbocyclyl is particularly preferred.
The term "carbocyclyl" designates a ring molecule, wherein the ring comprises
carbon atoms.
The ring may be formed exclusively by carbon atoms, but does not have to.
Accordingly,
one, two or more heteroatoms may be present. The ring may be saturated in
which case it
would be a cyclic alkane, optionally comprising one or more heteroatoms. The
ring may
contain one, two or more double bonds. If said double bonds are conjugated,
carbocyclyl is
an aryl moiety.
In a particularly preferred embodiment, (a) said carbocyclyl is aryl or
biaryl, aryl being
monocyclic or bicyclic and preferably phenyl or naphthyl; and (b) said
heterocarbocyclyl is
heteroaryl, heteroaryl being monocyclic or bicyclic, bi-heteroaryl, aryl
heteroaryl, heteroaryl
aryl or heterocycloalkyl, heteroaryl preferably being furyl, thienyl,
oxazolyl, isoxazolyl,
pyrazolyl, imidazolyl, thiazolyl, pyridinyl, pyrazinyl, pyridazinyl or
quinolinyl, aryl preferably
being phenyl, heterocycloalkyl preferably being morpholinyl or
tetrahydrofuranyl.
Particularly preferred is that heterocarbocyclyl is heteroaryl, in particular
monocyclic
heteroaryl.
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In a particularly preferred embodiment R11 is selected from 2-methyl thiazol-5-
y1; 4-
morpholinyl methyl; 1,4-dichloro 2-phenyl; 6-phenylpyridin-2-y1; pyrazin-2-y1;
3-furyl; 2-thienyl;
5-oxazoly1; 5-isoxazoly1; (5-Me)-3-isoxazoly1; (5-iPr)-3-isoxazoly1; (5-
MeOCH2)-3-isoxazoly1;
3-pyrazoly1; 2-imidazoly1; (N-Me)-3-pyrazoly1; (N-Me)-2-imidazoly1; (5-Me)-3-
pyrazoly1; 4-
pyridinyl; 4-pyridazinyl; 2-(R)-tetrahydrofuranyl; 2-(S)-tetrahydrofuranyl; (5-
Me)-3-isoxazolyl-
NH-; pyrazin-2-y1; naphth-2-y1; quinolin-2-y1; 4-biphenyl; 3-biphenyl; 4-
phenylpyridin-2-y1; 3-
phenyl-pyridin-2-y1; 5-phenylpyrazin-2-y1; 6-phenylpyrazin-2-y1; 2-phenyl-
thiazol-4-y1; and 5-
R111-isoxazol-3-y1;
wherein RII' is selected from methyl, 4-morpholinyl methyl, 1,2,4-triazoly1
methyl, imidazolyl
methyl and N-methyl piperazinyl methyl.
Especially preferred is that R11 is 2-methyl thiazol-5-yl.
Especially preferred compounds of the first aspect are the compounds of
formulae (11a) and
(11b):
0
0 0
0
R
R 1 ).HO
0
(11a) (11b),
wherein R1 is R11-CO-Ser(OMe)-Ser(OMe)-NH-CH(CH2-C6H5), R11 being as defined
above,
and wherein most preferably R1 is 2-methyl thiazol-5-y1 carbonyl Ser(OMe)-
Ser(OMe)-NH-
CH(CH2-C6H5).
In a second aspect, the present invention provides the use of a compound as
defined in the
first aspect as a proteasome inhibitor.
One or a plurality of compounds in accordance with the first aspect may be
used.
Preferably, said proteasome is a proteasome core particle (CP) which is also
known as 20S
proteasome. In a preferred embodiment, the constitutive core particle (cCP) is
used.
Alternatively, tissue-specific proteasomal subtypes such as immunoproteasome
(iCP) or the
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thymusproteasome (tCP) may be used.
A proteasome inhibitor in accordance with the present invention is a compound
which inhibits
one or more proteolytic activities of a proteasome. A proteasome comprises a
plurality of
proteolytic sites. Preferably, said proteolytic sites are as follows: a site
with caspase-like
proteolytic activity, a site with trypsin-like proteolytic activity and a site
with chymotrypsin-like
proteolytic activity. Proteasome inhibitors in accordance with the present
invention are
capable of inhibiting at least one of these sites. Preferred are compounds
which are capable
of inhibiting two or all three of these sites.
Related thereto, the present invention provides, in a third aspect, a method
of inhibiting a
proteasome, said method comprising bringing into contact a proteasome and a
compound as
defined in the first aspect, provided that methods for treatment of the human
or animal body
by therapy and diagnostic methods practised on the human or animal body are
excluded
and/or said method is performed in vitro or ex vivo.
Also provided is a method of inhibiting a proteasome, said method comprising
bringing into
contact a proteasome and a compound as defined in the first aspect. The method
may be
performed in vivo.
It is understood that said bringing into contact is effected under conditions
which allow a
physical contact between said compound and the proteasome. Suitable conditions
include
aqueous solutions, such as buffered aqueous solutions. Exemplary conditions
can be found
in the examples enclosed herewith.
The examples include also a proteasome activity assays in accordance with the
invention.
A preferred proteasome activity assay is as follows. The activity assay is
based on the
cleavage of 7-amino-4-methyl-coumarin (AMC) substrates consisting of the
fluorophore AMC
fused N-terminal to a peptide backbone. These substrates are fluorogenic as
the cleaved
AMC exhibits fluorescence (emission wavelength: 380 nm; emission wavelength:
460 nm).
For every active site a specific substrate is used (LLVY-AMC for the
chymotryptic-like site,
LLE-AMC for the caspase-like site, and RLR-AMC for the tryptic-like site).
Proteasome
activity is monitored by first mixing inhibitor and substrate simultaneously
in assay buffer and
incubation for 3 min at 37C . After the addition of 50 nM proteasome and
mixing, the
increase in fluorescence (excitation wavelength: 380 nm; emission wavelength:
460 nm) is
monitored with a spectrofluorimeter. DMSO concentration is kept
for all measurements.
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The fluorescence readout correlates with proteasome activity. This procedure
is utilized to
determine the first-order rate constant of inhibition. The IC50, which refers
to the inhibitor
concentration needed to decrease enzyme activity by 50%, can be determined by
measuring
proteasome activity at varying inhibitor concentrations.
In a fourth aspect, the present invention provides a medicament or lead
compound for
developing a medicament comprising or consisting of a compound as defined in
the first
aspect.
The terms "medicament" and "pharmaceutical composition" are used equivalently
herein. A
pharmaceutical composition in accordance with the present invention may
comprise a
pharmaceutically acceptable carrier, diluent or excipient.
Examples of suitable pharmaceutically acceptable carriers, excipients and/or
diluents are
well known in the art and include phosphate buffered saline solutions, water,
emulsions, such
as oil/water emulsions, various types of wetting agents, sterile solutions
etc. Compositions
comprising such carriers can be formulated by well known conventional methods.
These
pharmaceutical compositions can be administered to the subject at a suitable
dose.
Administration of the suitable compositions may be effected by different ways,
e.g., by
intravenous, intraperitoneal, subcutaneous, intramuscular, topical,
intradermal, intranasal or
intrabronchial administration. It is particularly preferred that said
administration is carried out
by injection. The compositions may also be administered directly to the target
site, e.g., by
biolistic delivery to an external or internal target site. The dosage regimen
will be determined
by the attending physician and clinical factors. As is well known in the
medical arts, dosages
for any one patient depends upon many factors, including the patient's size,
body surface
area, age, the particular compound to be administered, sex, time and route of
administration,
general health, and other drugs being administered concurrently.
Pharmaceutically active
matter may be present in amounts between 1 ng and 10 mg/kg body weight per
dose;
however, doses below or above this exemplary range are envisioned, especially
considering
the aforementioned factors. If the regimen is a continuous infusion, it should
also be in the
range of 1 pg to 10 mg units per kilogram of body weight per minute. Preferred
doses are in
the range from about 10 to about 40 mg/m2 body surface, which correspond to
about 0.25 to
about 1 mg/kg body weight.
One or more compounds in accordance with the first aspect of the present
invention may be
the only pharmaceutically active agent(s) comprised in a medicament in
accordance with the
present invention. Alternatively, one, two or more further pharmaceutically
active agents may
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be present. Said further pharmaceutically active agents are preferably
selected from known
proteasome inhibitors and/or therapeutic agents for the treatment of multiple
myeloma.
Known proteasome inhibitors are discussed above and include bortezomib,
carfilzomib,
ixazomib, marizomib, oprozomib, delanzomib, epoxomicin, dihydroeponemycin.
Exemplary
agents for the treatment of multiple myeloma are lenalidomide and
dexamethasone as well
as the combination of the latter two.
The development of a lead compound into a medicament is also known as lead
optimization.
Methods for the optimization of the pharmacological properties of lead
compounds are
known in the art and comprise a method of modifying a lead compound to
achieve: (i)
modified site of action, spectrum of activity, organ specificity, and/or (ii)
improved potency,
and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv)
decreased side
effects, and/or (v) modified onset of therapeutic action, duration of effect,
and/or (vi) modified
pharmacokinetic parameters (resorption, distribution, metabolism and
excretion), and/or (vii)
modified physico-chemical parameters (solubility, hygroscopicity, color,
taste, odor, stability,
state), and/or (viii) improved general specificity, organ/tissue specificity,
and/or (ix) optimized
application form and route by (i) esterification of carboxyl groups, or (ii)
esterification of
hydroxyl groups with carboxylic acids, or (iii) esterification of hydroxyl
groups to, e.g.
phosphates, pyrophosphates or sulfates or hemi-succinates, or (iv) formation
of
pharmaceutically acceptable salts, or (v) formation of pharmaceutically
acceptable
complexes, or (vi) synthesis of pharmacologically active polymers, or (vii)
introduction of
hydrophilic moieties, or (viii) introduction/exchange of substituents on
aromates or side
chains, change of substituent pattern, or (ix) modification by introduction of
isosteric or
bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi)
introduction of
branched side chains, or (xii) conversion of alkyl substituents to cyclic
analogues, or (xiii)
derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation
to amides,
phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi)
transformation of
ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters,
oxazolidines,
.. thiazolidines or combinations thereof.
The various steps recited above are generally known in the art. They include
or rely on
quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch-
Analysis and
Related Approaches", VCH Verlag, Weinheim, 1992), combinatorial biochemistry,
classical
chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche
Apotheker
Zeitung 140(8), 813-823, 2000).
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In a fifth aspect, the present invention provides a compound as defined in the
first aspect for
use in a method of treating, ameliorating or preventing cancer, an autoimmune
disease,
muscular dystrophy, emphysema, or cachexia accompanying cancer or AIDS.
More generally speaking, the compounds in accordance with the invention are
useful in
methods of treating, ameliorating or preventing any condition which is
amenable to
treatment, prevention or amelioration via inhibition of a proteasome.
In a preferred embodiment, said cancer is a lymphoid malignancy, preferably
selected from
multiple myeloma (MM) including relapsed and refractory MM; non-Hodgkin
lymphoma such
as B-cell lymphomas including mantle cell lymphoma (MCL) and diffuse large B-
cell
lymphoma (DLBCL), and Waldenstrom macroglobulinaemia.
In a further preferred embodiment said autoimmune disease is rheumatoid
arthritis, systemic
lupus erythematosus, Sjorgen's syndrome or scleroderma.
In a sixth aspect, the present invention provides a method of identifying a
compound capable
of inhibiting a proteasome, said method comprising bringing into contact a
test compound of
formula (III)
R2
X -
R4 Y Z
R3
(III),
wherein R4 is an organic group, and X, Y, Z, R2 and R3 are as defined in
accordance with the
first aspect or preferred embodiments thereof,
with a proteasome, wherein a decreased activity of the proteasome in presence
of said test
compound as compared to the absence thereof is indicative of said test
compound being a
compound capable of inhibiting a proteasome.
This aspect relates to a screening method. Screening may be a biochemical
screen or a
cellular screen. Generally speaking, a variety of assay designs are available
for the purpose
of identifying compounds which are capable of inhibiting the activity of a
given target
molecule, here a proteasome. One of the established distinctions is between
cellular assays
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and biochemical assays. Cellular assays are sometimes viewed as mimicking
closer the in
vivo situation, however, they suffer from the drawback that any candidate
compound
generally has to pass the cell membrane in a first step. Biochemical assays
are simpler in
that respect. The target, here a proteasome, may be presented in enriched or
purified form in
aqueous solution. In such an assay scenario there is no membrane barrier. The
conditions,
though, may be further remote from the environment in the organism to be
subjected to
therapy.
A negative control for the screen is the absence of any test compound as
disclosed above.
As positive controls one or more compounds in accordance with the first aspect
or known
proteasome inhibitors as disclosed herein may be used.
As known in the art, screening methods may be implemented in a high throughput
fashion.
Accordingly, said method may be effect in a high throughput format. High
throughput assays
generally may be performed in wells of microtiter plates, wherein each plate
may contain 96,
384 or 1,536 wells. Handling of the plates, including incubation at
temperatures other than
ambient temperature, and bringing into contact of these compounds with the
assay mixture is
preferably effected by one or more computer controlled robotic systems
including pipetting
devices. In case large libraries of test compounds are to be screened and/or
screening is to
be effected within short time, mixture of, for example 10, 20, 30, 40, 50 or
100 test
compounds may be added to each well. In case a well exhibits biological
activity, in the
present case inhibition of a proteasome, said mixture of test compounds may be
de-
convoluted to identify one or more test compounds in said mixture giving rise
to said activity.
A compound capable of inhibiting a proteasome is also referred to as
proteasome inhibitor.
Inhibition preferably amounts a decrease of activity of at least 10%, at least
20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or
at least 90%.
Further preferred is a 102-fold, 103-fold, 104-fold, 105-fold, 108-fold, 107-
fold, 108-fold or 109-
fold reduction of activity.
An art-established measure of inhibitory activity is the IC50 value which is
the inhibitor
concentration where 50% inhibition occurs. Preferably, proteasome inhibitors
to be identified
by the method of identifying of the present invention as well as compounds in
accordance
with the first aspect exhibit IC50 values in the one digit pM range,
preferably below 1 pM,
more preferably below 100 nM, below 10 nM, below 1 nM, or below 100 pM. IC50
values are
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preferably determined using the proteasome activity assay as described above.
In accordance with the method of the sixth aspect, R4 is not particularly
limited. In fact, one of
the applications of this method is the identification of further targeting
moieties which
targeting moieties may be different from the targeting moieties defined in
relation to the first
aspect of the invention.
As noted above, R4 is an organic group. It may have a molecular weight between
200 and
1,000 Da.
In a preferred embodiment, said activity is a proteolytic activity, preferably
selected from
caspase-like, trypsin-like and chymotrypsin-like activity.
In a further preferred embodiment said proteasome is comprised in an in vitro
or ex vivo cell.
This preferred embodiment refers to a cellular assay.
A preferred cellular assay is as follows. Cells (e.g. HEK 293) are transfected
with the
pZsProSensor-1 vector (Clontech Laboratories, Inc.), a reporter gene construct
for
proteasomal activity. This vector encodes for a destabilized green fluorescent
protein variant
(ZsGreen) fused C-terminally to a specific degradation motif mediating the
recognition and
degradation by the proteasome. The fluorescence readout (excitation
wavelength: 493 nm;
emission wavelength: 505 nm) of expressed ZsGreen is used to monitor
proteasome activity.
Inhibition of the proteasome causes accumulation of ZsGreen correlating with
an increased
fluorescence signal in the GFP channel.
In a seventh aspect, the present invention provides a compound of formula (IV)
R2
X
Z
R3
(IV),
wherein
A is selected from NH-NH2, N3 and a click chemistry functional group;
X, Y, Z, R2 and R3 are as defined above.
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In an eighth aspect, the present invention provides the use of a compound of
formula (IV)
R2
X
Z
R3
(IV),
wherein
A is selected from NH-N H2, N3 and a click chemistry functional group;
X, Y, Z, R2 and R3 are as defined above;
in the synthesis of a proteasome inhibitor, of a pharmaceutically active agent
or of a lead
compound for the development of a pharmaceutically active agent, said
pharmaceutically
active agent preferably being for use in a method of treating or preventing
cancer or an
autoimmune disease.
The latter two aspects of the invention are directed to the headgroup or
warhead of
proteasome inhibitors in accordance with the present invention.
Generally speaking, A is a reactive group. Art-established reactive groups may
be used. The
purpose of A is to provide robust irreversible coupling to targeting moieties
such as R1 as
defined above. The reactive group A may be used for coupling to any targeting
moiety. Upon
coupling to a targeting moiety, compounds in accordance with the first aspect
may be
obtained.
"Click-chemistry is an art-established term; see e.g. Kolb et al. (2001) Click
chemistry:
diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40
(11):2004;
Sletten et al. (2009) Bioorthogonal Chemistry: Fishing for Selectivity in a
Sea of Functionality.
Angew. Chem. Int. Ed.48:6998; Jewett et al.(2010) Cu-free click cycloaddition
reactions in
chemical biology. Chem. Soc. Rev. 39(4):1272; Best et al. (2009) Click
Chemistry and
Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of
Biological Molecules.
Biochemistry.48:6571; and Lallana et al. (2011) Reliable and Efficient
Procedures for the
Conjugation of Biomolecules through Huisgen Azide¨Alkyne Cycloadditions.
Angew. Chem.
Int. Ed. 50:8794.
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As regards the embodiments characterized in this specification, in particular
in the claims, it
is intended that each embodiment mentioned in a dependent claim is combined
with each
embodiment of each claim (independent or dependent) said dependent claim
depends from.
For example, in case of an independent claim 1 reciting 3 alternatives A, B
and C, a
dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending
from claims 1
and 2 and reciting 3 alternatives G, H and I, it is to be understood that the
specification
unambiguously discloses embodiments corresponding to combinations A, D, G; A,
D, H; A,
D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H;
B, D, I; B, E, G; B, E,
H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C,
E, H; C, E, I; C, F, G;
C, F, H; C, F, I, unless specifically mentioned otherwise.
Similarly, and also in those cases where independent and/or dependent claims
do not recite
alternatives, it is understood that if dependent claims refer back to a
plurality of preceding
claims, any combination of subject-matter covered thereby is considered to be
explicitly
disclosed. For example, in case of an independent claim 1, a dependent claim 2
referring
back to claim 1, and a dependent claim 3 referring back to both claims 2 and
1, it follows that
the combination of the subject-matter of claims 3 and 1 is clearly and
unambiguously
disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In
case a further
dependent claim 4 is present which refers to any one of claims 1 to 3, it
follows that the
combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of
claims 4, 3 and
1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.
The figures show:
Figure 1: Chemical structures of representative members of the epoxyketone
class of
proteasome inhibitors. Shown are the natural products epoxomicin (top right)
and
dihydroeponemycin (bottom right), which were isolated from bacterial strains.
Also depicted
are synthetic derivative of the parent natural product molecules. Carfilzomib
(top left) was
recently approved for the treatement of multiple myeloma and is currently in
clinical trials for
the treatment of other cancers. Oprozomib is currently in clinical trials as
an orally available
multi-potent inhibitor for the treatment of several cancers.
Figure 2: Crystal structures of co-crystal structures of human 20S proteasomes
in complex
.. with epoxyketone inhibitors. 2Fo-Fc electron density maps are shown
contoured at 1.5 a for
the proteasome active site inhibited with carfilzomib (top left), oprozomib
(bottom left),
epoxomicin (top right) and dihydroeponemycin (bottom right). Note that the
electron density
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map reveals density for an additional carbon atom. In all cases, the modeled
cyclic ring
structure is consistent with a 1,4-oxazepane structure formed by the reaction
of epoxyketone
inhibitors with the active site catalytic threonine side chain.
Figure 3: Confirmation that a 7-membered 1,4-oxazepane ring structure is
formed upon
inhibition of proteasomes with epoxyketone inhibitors. Shown are the chemical
structures of
Oprozomib (top left) and dihydroeponemycin (top right). The oval highlights
that Oprozomib
contains a methyl group and dihydroeponemycin a methanolic group in the carbon
atom in
a-position to the ketone, respectively. The bottom panels illustrate the 135
active site inhibited
with Oprozomib (bottom left) and Dihydroeponemycin (bottom right), along with
an omit map
contoured at 4a for the inhibitor, the cyclic linkage and 135Thr2. The main
chain segments of
135 residues 2, 19-21, 33, 45-50, 129-131, 169,170 and 136 125,126 are
indicated along with
the 135 side chains of Thr2, Thr23, Lys33, Ser130 and the side chains of 136
Asp125, Pro126
as sticks. Dashed lines signify hydrogen bonds
3.2 A distance). In the respective bottom
right panels close-up views of the inhibitor-Thr1 linkages are shown along
with an omit map
contoured at 6a. Note that the electron density map does not support a chiral
center in the
case of epoxomicin, which contains a methyl and a methanolic group, which
would be a
consequence of the proposed mechanism for 1,4-morpholine ring formation
(bottom left)
(Groll et al., /oc. cit.). The dihydroeponemycin structure does not reveal
electron density
consistent with the presence of two methanolic groups, yet again disproving
1,4-morpholine
ring formation (bottom right).
Figure 4: Attempts to model a 1,4-morpholine ring structure in the linkage
occurring between
epoxyketone inhibitor and the proteasome active site Threonine1. Shown is an
overlay of the
proteasome active site (main chain) inhibited by a 1,4-oxazepane linkage and
the attempted
1,4-morpholine linkage. The attempted 1,4-morpholine refinement reveals that a
van-der-
Waals clash occurs with the methyl group of the inhibitor and the main chain
carbonyl atoms
of R19 and Y169. Additionally, strong negative difference density at 5 sigma
is visible at the
C5 methanol atom of the 1,4-morpholine linkage. Positive densities are visible
at positions 4
and 5 of the 1,4-oxazepane linkage. These findings entirely exclude formation
of 1,4-
morpholine ring formation in the epoxyketone inhibited proteasome active site.
Figure 5: Enzymatic characterization of proteasome catalytic activity.
Measurements were
performed as described in methods. The left panel depicts a typical kinetic
experiment,
where the increase in fluorescence signal of the AMC released by proteolytic
cleavage is
plotted against time. The left window signifies an activation phase in
enzymatic peptide
cleavage, whereas the right window represents the steady-state phase. In the
right panel the
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PCT/EP2017/063699
specific activities for the human 20S proteasome purified by our method are
indicated for the
pre-steady-state (prior to activation) or and the steady-state phases,
respectively. The
equation shown in the right panel was used to perform fits against the
experimental data. Fo
designates the initial fluorescence, AFss: fluorescence increase in the steady-
state part of the
measurement, kact: the rate constant. The exponential term with the rate
constant kact is used
to describe the activation phase of the reaction.
The examples illustrate the invention.
Example 1
Crystallography
Initial phases for human 20S proteasomes were determined by molecular
replacement using
the murine 20S structure (PDB ID: 3UNE). The model was then optimized by
several rounds
of interactive manual model building in Coot and refinement in Refmac5. The
obtained
structures display excellent stereochemistry with typical values for Rwork=
18% and Rfree=
21%, reveal superb electron densities for all 6724 residues and reveal several
ligands as
present in buffers used for purification and crystallization.
Using a dataset to 1.8 A resolution, we created a reference model. With the
availability of this
excellent model for the human 20S proteasome, now structure determination
takes minutes
by automated refinement of the reference model against integrated and scaled X-
ray data
from related crystals. Bound ligands can then be rapidly identified in
difference density maps
and modeled interactively in Coot.
Native crystals were soaked with the epoxyketone inhibitors shown in Figure 1.
Co-crystal
structures of human 20S proteasomes in complex with these inhibitors at
resolutions
between 1.9 ¨ 2.1 A (0.3-0.5 A better resolution than presently available
structures) were
solved. Surprisingly, after refinement of these co-crystal structures, which
allow the modelling
of all parts of human 20S proteasomes at atomic resolution, it was not
possible to visualize
the presumed 1,4-morpholine ring structure in the inhibited state. The
electron density maps
in the new epoxyketone/human 20S proteasome co-crystal structures in all cases
did not
agree with the formation of a 1,4-morpholine 6-ring. Instead, density for an
additional atom
became clearly visible, which is consistent with a 7-ring structure. In fact,
modelling the cyclic
molecule visible in the inhibited state formed by the reaction of the
epoxyketone inhibitors
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PCT/EP2017/063699
with the active site catalytic threonine amino acid revealed that it
represents a 1,4-oxazepane
7-ring structure (Figure 2).
The observation of a 1,4-oxazepane structure contrasts with previously
presumed chemical
inhibition mechanisms of 20S proteasomes by epoxyketones. Therefore, a control
experiment was performed to ensure that the observation of this 7-membered
ring structure
is true and the modelling of the 1,4-oxazepane structure in the inhibited
proteasome active
site is justified. Additionally, this control experiment should provide
insight into the chemical
inhibition mechanism by which the 7-ring structure is formed upon proteasome
inhibition. For
this control experiment, the co-crystal structures determined using the
epoxyketone inhibitors
epoxomicin and dihydroeponemycin were compared. Epoxomicin contains an epoxide
group
with a methyl ligand at the carbon atom, where the nucleophilic attack is
presumed to occur
in order to form the presumed 1,4-morpholine inhibited ring structure (Figure
3, top left).
Additionally, after formation of the presumed 1,4- morpholine structure ring
opening of the
epoxide at the carbon atom in a-position to the ketone should yield a stereo
center, which
contains both a methyl and a methanolic group (Figure 3, right panel) (Groll
et al., /oc. cit.).
Dihydroeponemycin in contrast already contains a methanolic ligand at the
carbon atom,
where the nucleophilic attack is presumed to occur in order to form the
proposed
1,4-morpholine inhibited ring structure (Figure 3, top right). As a
consequence, after
formation of the proposed 1,4-morpholine structure ring opening of the epoxide
at the carbon
atom in a-position to the ketone should yield a non-chiral center, which
contains two
methanolic groups. At the resolutions at which both co-crystal structures were
determined
(1.9 and 2.0 A), it is possible to verify if this is the case. Surprisingly
however, the electron
density maps of both structures revealed that the inhibited state is formed by
the covalent
attack of the electrophilic carbon atom in 13-position to the ketone (Figure
3, bottom panels),
excluding the possibility that a 1,4-morpholine ring structure is formed in
the inhibited state
by the absence of electron density compatible with a methanolic group in the
case of
epoxomicin and two methanolic groups in dihydroeponemycin.
Attempts to model the electron density in the active site of the
dihydroeponemycin complex
by a 1,4-morpholine ring structure linkage were unsuccessful (Figure 4). 1,4-
morpholine
linkage refinement resulted in a severely distorted molecular geometry
characterized by the
elongation of the N4-carbon bonds by 0.1-0.2 A, shortening of the C5-alcohol
carbon bond
by 0.1 A and deviation of the methyl-05-alcohol bond angle by -20 degrees off
the expected
value. Additionally, the methyl carbon exhibited a van-der-Waals distance of
3.0 A to the
Arginine 19 and Tyrosine 169 main chain oxygen atoms, which is too close.
Moreover, strong
negative difference density peaks in difference maps contoured at 5 sigma
levels at the C5
,
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methanol oxygen of the 1,4-morpholine ring model, as well as positive density
peaks
contoured at 4.5 sigma levels close to positions 4 and 5 of our 1,4-oxazepane
ring model
remained after this 1,4-morpholine ring refinement. In contrast, no residual
difference (neither
negative nor positive) density was present in the refined 1,4-oxazepane
linkage in density
maps contoured above 2.3 sigma.
Example 2
Synthesis
,
,
,
.
0 0
,
>QLOel
HN OH ____
1. EDC, DMAP, 9--0)FIN 0 * Mel, --)----di'L FIN
0 *
(1) 0 MeIdrum acid o 0 K2CO3 o 0
2.BnOH (2) (3)
.
,
TEA,
'
CH2Cl2
.
,
0 0
o o o 0
H H
\ AN
, _____________________________________________________________ H2N
0
NJL 0 , ___________ N 0 a
HOBt
N 0 -,.., 0 Methal101 N 0 -, 0
EDC, o o
(6) ? (5) ?
peptide (18) (4)
OH 0
N-Hydroy- i.
succinimide,
EDC
µ'
I
0 o
syt,) jy Li li o,IA
---i 1 11 ---:--"-N
r H
(7) ?
Scheme 3: Synthesis of NHS-ester. Details are given below; see steps A to F.
0 0,c,"' '
1 _ 1 il
9'-'051'HN __ OH
MeNHOMe, Ha ...)---01HN N' __________
o
0 NEt3, BOB, CH2Cl2 o i o 0-
-/ K2CO3 0 0-1
(1) (8) (8) (9-1)
ITFA,
CH2Cl2
0 i
o
o o o
fyi jc)
N t,
1,
./
____eyLEXNHAN ,0 , 0\
MeCOH, /YL HI N N 0 \ EH (1::: II H
2 N
, H r H
Isl- 0 ,-.7 0 p-TSOH
,___,s o -,...,
7 (12) (11) ? peptide (18)
(10)
Scheme 4: Synthsesis of p-keto aldehyde. Details are given below; see steps G
to K.
,
,
,
,
,
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30
PCT/EP2017/063699
0 0
>L0j1,) N,c0H >LoiNfy0 ___________ fy0
T, DMAP, TFA, CH2Cl2 H2NEA HOBt/HBTU,
o Benzyl chloro- 0 0 DIEA 0
(13) formate (14) (15) peptide (13) (16)
TFA,
CH2Cl2
0
OH
NifrN--- -0 H2NNOio
0 7,0 H2, Pd/C, o HOBt/HBTU, 0
0
(19) (18)
Methanol DIEA (17)
I ?
Nrq
co2H
Scheme 5: Synthesis of tripeptide. Details are given below; see steps L to Q.
A) To a solution of N-Boc-L-Phenylalanine (1) in CH2Cl2 is added a EDC (1
eq.), DMAP (1
eq.) and Meldrum's acid (1 eq.). The reaction is stirred at room temperature
for 17 hours,
then poured into 1 M HCl. The layers are separated and the aqueous layer is
extracted three
times with CH2Cl2. The organic layers are combined, washed with brine, dried
over MgSO4
and concentrated in vacuum. The residue is heated to 80 C in toluene and
after the addition
of benzyl alcohol (1 eq.) for 4 hours. The solvent is the removed in vacuum
and the residue
purified by flash chromatography and elution with ethyl acetate to yield (2).
B) A solution of (2), methyl iodide (3 eq.) and potassium carbonate (2 eq.) in
acetone is
heated under reflux for 17 hours. 2 volume equivalents of water are added and
the resulting
mixture extracted three times with 2 volume equivalents ethyl acetate. The
organics are
combined, dried over MgSO4 and concentrated in vacuum. The residue is purified
by
preparative HPLC in 0.1 % formic acid in water, with a gradient to 0.1 %
formic acid in
acetonitrile to give (3).
C) A solution of (3) is stirred in a 10 % TFA solution in CH2Cl2 for 17 hours
at room
temperature. The solvent is subsequently removed in vacuum to yield (4).
D) To (4) (1 eq.) in CH2Cl2 and triethylamine (0.001 eq.) is added (19) (1
eq.), HOBT (0.2 eq.)
and EDC (2 eq.). The solution is stirred at 25 C for 24 hours and then washed
three times
with saturated sodium hydrogencarbonate solution, once with deionized water
and brine
each, and the organic layer is dried over MgSO4. The solvent is removed in
vacuum and the
residue purified by flash chromatography eluting with 1:1 ethyl acetate:n-
hexane to yield (5).
E) (5) is stirred in methanol containing 10% Pd/C under a hydrogen atmosphere
(1 atm).
After 2 hours the mixture is filtered through celite and the solvent removed
in vacuo to the
product (6).
F) To (6) in CH2Cl2 is added, EDC (2 eq.) and N-Hydroxysuccinimide (2 eq.) and
the mixture
stirred for 2 hours. The solvent is then removed in vacuo and the residue
purified by flash
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PCT/EP2017/063699
chromatography, eluting with 1:5 ethyl acetate:n-hexane to yield (7).
G) To a stirred solution of Boc-L-Phenylalanine in CH2C12 is added 0,N-
dimethylhydroxylamine hydrochloride (1 eq.), triethylamien (2 eq.) and BOP (1
eq.). After 3.5
hours the solution is diluted 4-fold with CH2C12 and washed three times with 3
M HCL, three
times with saturated sodium hydrogencarbonate and three times with brine. The
organic
layer is then dried over MgSO4, the solvent removed in vacuo and the residue
purified by
flash chromatography with 1:3 ethyl acetate:n-hexane to yield (8).
H) The Weinreb amide (8) in THF under an Argon atmosphere is cooled to 0 C
and 1,3-
Dioxacyclopenty1-2-MgBromide (5 eq.) in THF added dropwise. The reaction is
allowed to
reach 25 C and after 4 hours of stirring, is quenched with 1 M HCl forming a
precipitate. The
precipitate is removed by filtration and washed three times with ethyl
acetate. The combined
organics are then washed with brine, dried over MgSO4 and the solvent removed
in vacuum.
The residue is then purified by flash chromatography using 1:4 ethyl acetate:
n-hexane as an
eluent to yield (9).
1) A solution of (9) is stirred in a 10 % TFA solution in CH2C12 for 17 hours
at room
temperature. The solvent is subsequently removed in vacuum to yield (10).
J) To (10) (1 eq.) in CH2C12 and triethylamine (0.001 eq.) is added (19) (1
eq.), HOBT (0.2
eq.) and EDC (2 eq.). The solution is stirred at 25 C for 24 hours and then
washed three
times with saturated sodium hydrogencarbonate solution, once with deionized
water and
brine each, and the organic layer is dried over MgSO4. The solvent is removed
in vacuo and
the residue purified by flash chromatography eluting with 1:1 ethyl acetate:n-
hexane to yield
(11).
K) A solution of (11) and p-Ts0H (0.1 eq.) in acetaldehyde (0.5 eq.) is
stirred under an argon
atmosphere at 15 C for 23 hours. The solvent is then removed in vacuum and
the residue
purified by flash chromatography, eluting with 1:5 ethyl acetate:n-hexane to
yield (12).
L) To a solution of Boc-methylserine (13) in DCM (Dichlormethane) TEA
(Triethylamine) and
DMAP (4-Dimethylaminopyridine) are added. The resulting solution is cooled to -
5 C, and
benzyl chloroformate is then slowly added via an addition funnel under an
atmosphere of
argon. The reaction is kept at the same temperature for 3 h and then diluted
with brine. The
layers are separated, and the aqueous layer is extracted with DCM. The organic
layers are
combined and dried over Na2SO4. The Na2SO4 is removed by filtration, and the
volatiles
are removed under reduced pressure. The resulting residue is purified by flash
chromatography using a mixture of hexane and ethyl acetate to provide
intermediate (14) as
white solid.
M) To a 0 C solution of intermediate (14) in DCM TFA (Trifluoroacetic acid)
is added slowly
via a funnel. The reaction is kept at the same temperature for 1 h,
concentrated, and dried
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PCT/EP2017/063699
under high vacuum overnight. The resulting residual TFA salt (15) is used in
the next step
without further purification.
N) To a -5 C mixture of aforementioned TFA salt (15), Boc-methylserine (13),
HOBt
(Hydroxybenzotriazole), and HBTU (2-(1H-benzotriazol-1-y1)-1,1,3,3-
tetramethyluronium
hexafluorophosphate) in THF (Tetrahydrofuran) (600 mL) is added DIEA slowly
via an
addition funnel. The reaction is kept at the same temperature for 4 h,
followed by dilution with
Et0Ac (Ethylacetate) and brine. The layers are separated, and the aqueous
layer is
extracted with Et0Ac (2 x 300 mL). The organic layers are combined and dried
over
Na2SO4. The Na2SO4 is removed by filtration, and the volatiles are removed
under reduced
pressure. The resulting residue is purified by flash chromatography using a
mixture of
hexane and ethyl acetate to provide dipeptide (16) as white solid.
0) To a 0 C solution of aforementioned intermediate (16) in DCM was added TFA
slowly via
an addition funnel. The reaction is kept at the same temperature for 2 h,
concentrated, and
dried under high vacuum overnight. The resulting residual TFA salt (17) is
used in the next
step without further purification.
P) To a -5 C mixture of TFA salt (17), 2-methylthiazole-5- carboxylic acid,
HOBt, and HBTU
in THF is added DIEA slowly. The reaction is kept at the same temperature for
4 h and then
diluted with Et0Ac and brine. The layers are separated, and the aqueous layer
is extracted
with Et0Ac. The organic layers are combined and dried over Na2SO4. The Na2SO4
is
removed by filtration, and the volatiles are removed under reduced pressure.
The resulting
residue is purified by flash chromatography using a mixture of hexane and
ethyl acetate to
provide benzyl ester (18) as white solid.
Q) (18) is stirred in methanol containing 10% Pd/C under a hydrogen atmosphere
(1 atm).
After 2 hours the mixture is filtered through celite and the solvent removed
in vacuum to the
product (19).
Example 3
Proteasome assays
Activity measurement
Preferred in vitro Assay.
All kinetic measurements were performed using a FluoroMax0-4 fluorescence
spectrophotometer (Horiba Scientific). Succinyl-Leucine-Leucine-Valine-
Tyrosine-7-amido-4-
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methylcoumarin (Suc-LLVY-AMC, Bachem) was used as substrate to determine the
chymotryptic-like activity of the 135 catalytic active site of the human 20S
proteasome (R. L.
Stein, F. Melandri, L. Dick, Kinetic characterization of the chymotryptic
activity of the 20S
proteasome. Biochemistry 35, 3899-3908 (1996)). The fluorescence emission of
hydrolyzed
AMC was continuously monitored at 460 nm (Aex = 380 nM). The reaction
temperature was
kept at 37 C for all measurements and the reaction buffer for enzymatic assays
specified in
Table S1 was used. Suc-LLVY-AMC and inhibitors (such as Oprozomib,
Dihydroeponemycin, Z-LLY-Ketoaldehyde; "Sue" designating Succinyl and "Z"
designating
Benzyloxycarbonyl) were dissolved in DMSO and stored at -80 C until usage.
The DMSO
concentration did not exceed 2% (v/v) in any measurement.For kinetic
characterization of
Suc-LLVY-AMC conversion, 0.035 mg/mL (50 nM) human 20S proteasome in reaction
buffer
was pre-incubated for 3 minutes at 37 C. The reaction was started by the
addition of
substrate and the fluorescence signal was measured continuously. For
determination of the
first-order rate constant of inhibition of the respective inhibitors, the
reaction mixture
containing reaction buffer, 150 pM substrate and either Oprozomib (50 pM),
Dihydroeponemycin (50 pM) or Z-LLY-Ketoaldehyde (15 pM) were pre-incubated at
37 C for
3 minutes. The reaction was then started by the addition of human 20S
proteasome to a final
concentration 50 nM. The fluorescence signal was measured continuously.
Data were analyzed and fitted with OriginPro 9.1 and KaleidaGraph 4.03. The
equation
shown in Figure 5 was used to analyze the chymotryptic-like catalytic activity
and catalytic
activation of the 20S proteasome. For the determination of the first-order
inactivation rate
constants, equations were used that contained either two exponential terms in
case of Z-
LLY-Ketoaldehyde, or two exponential terms plus a linear term for epoxyketones
(Oprozomib
and Dihydroeponemycin). The first of the two exponential terms accounts for
the catalytic
activation, whereas the second exponential term represents the catalytic
inactivation by
inhibitory action. The linear term in case of the epoxyketones was used to
account for the
residual activity of the proteasome after inactivation.
Alternative kinetic assay.
Time point measurements of the activity assays are performed to acquire an
initial tendency
of inhibitor binding. Different concentrations of 20S proteasome are used for
each active site:
0.05 mg/ml for CL (Chymotryptic-like activity) and PGPH (Peptidyl-glutamyl
peptide
hydrolyzing activity) and 0.075 mg/ml for TL (Tryptic-like activity). The
final reaction volume is
30 p1/well. A total number of five repetitions are performed to obtain root
mean square
deviation (RMSD), including a blank and a 100% initial activity reaction. The
steps are as
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follows.
(1) A master mix is prepared: Respective amount proteasome (according to PGPH,
TL, or CL
activity determination).
(2) Eppendorf tubes are prepared with the amount of the respective inhibitor
to be analysed;
e.g. 500 pM concentration of the ligand in 30 pl is 1.5 pl per assay of a 10
mM inhibitor stock
solution.
(3) 28.5 pl of the master mix is added to each Eppendorf tube. This solution
is incubated for
min at room temperature and transferred to the respective wells of the 96 well
plates.
(4) Following incubation, 1 pl of a 7.5 mM stock solution of substrate for the
caspase,
10 chymotryptic or tryptic site is added, giving a final substrate
concentration of 250 pM. The
plate is centrifuged and incubated at RT for 1 h.
(5) 300 pl of buffer is added to the reaction and the remaining proteasome
activity is
subsequently recorded by fluorescence at Ex (Excitation wavelength) 360 nm -
Em
(Emission wavelength) 460 nm.
15 (6) The remaining activity is calculated using the blank and 100%
initial activity.
Once proteasome inhibition is observed through the time point measurements,
the half
maximal inhibitory concentration (IC50) measurements can be performed. The
percentage of
the remaining activities is then plotted against the log concentration of the
respective
inhibitor. The obtained data are fitted with a conventional statistical
program, for example as
described in: Groll M, Gallastegui N, Marechal X, et al (2010) 20S Proteasome
Inhibition:
Designing Non-Covalent Linear Peptide Mimics of the Natural Product TMC-95A.
ChemMedChem 5:1701-1705.
.. Reference for assay:
A description of an activity assay can be found in Gallastegui, N., & Groll,
M. (2012).
Analysing Properties of Proteasome Inhibitors Using Kinetic and X-Ray
Crystallographic
Studies. In Methods in Molecular Biology (Vol. 832, pp. 373-390).
doi:10.1007/978-1-61779-
474-2_26.
Cellular assays.
A further example is the Z-Sensor Proteasome assay from Takara/ Clontech.
ZsProSensor-1
is a proteasome-sensitive fluorescent reporter. It is a fusion of a bright
green fluorescent
protein (Exmax = 496 nm, Emmax = 506 nm) with a degradation domain which
targets the
protein for rapid degradation by the proteasome. The cells emit green
fluorescence when
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there is a drop in proteasome activity.
Alternative methods:
Proteasome-GloTM Chymotrypsin-Like, Trypsin-Like and Caspase-Like Cell-Based
Assays
(Promega).
GFP-Assay: Bence N, Bennett E, Kopito R, Deshaies R. Application and analysis
of the
GFP(u) family of ubiquitin-proteasome system reporters. Ubiquitin and Protein
Degradation,
Pt B. 2005;399:481-490.
A cellular assay for the immunoproteasome is the following:
Fluorogenic in vitro assay (Immunoproteasome): (Basler, M., & Groettrup, M.
(2012).
Immunoproteasome-Specific Inhibitors and Their Application. In Methods in
Molecular
Biology (Vol. 832, pp. 391-401). doi:10.1007/978-1-61779-474-2_27)
In order to test whether your IP inhibitor is cell permeable, the following
method based on
proteasome immuno- precipitation and in vitro activity assay can be used. The
steps are as
follows.
(1) Incubate cells for 2 h with desired concentration of IP inhibitors in cell
culture media at
37 C. We normally use mouse splenocytes (one spleen per sample). As control,
use an
equal number of cells without inhibitor.
(2) Wash cells three times with PBS to remove unbound inhibitor.
(3) Lyse cells in 500 pl lysis buffer and incubate for 20 min on ice.
(4) Centrifuge the lysates for 10 min at 20,800 x g to remove debris.
(5) Discard pellet and add 3 pl of polyclonal rabbit-anti-mouse proteasome
antibody and
50 pl protein A microbeads to the supernatant and incubate for 30 min on ice.
(6) Insert p column into magnet.
(7) Equilibrate p column with 1 ml NET-TON buffer.
(8) Load lysate on p column and discard flow through.
(9) Wash column twice with 1 ml NET-TON buffer and three times with NET-T
buffer.
.. (10) Add 50 pl of a fluorogenic substrate and incubate column for 30 min at
37 C.
(11) Add 200 pl lysis buffer and collect eluate.
(12) Measure the fluorescence in 100 pl of the eluate (96-well plate, flat
bottom, black). The
fluorescence in the eluate corresponds to the activity of the retained
proteasome in the
column.
LacZ assay (Immunoproteasome); see Basler, M., & Groettrup, M. (2012).
Immunoproteasome-Specific Inhibitors and Their Application. In Methods in
Molecular
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Biology (Vol. 832, pp. 391-401). doi:10.1007/978-1-61779-474-2_27.
Numerous MHC-I restricted CD8+ T-cell epitopes have been described to be
dependent on
IP subunits. Investigating the pro- cessing of such T-cell epitopes can test
specificity of IP
inhibitors. In order to analyse the LMP7-selective inhibitor PR-957, we
investigated the male
HY-derived CTL-epitope UTY 246-254, which was reported to be LMP7 dependent.
Therefore, we treated male splenocytes with PR-957 and detected MHC-I
presented UTY
246-254 peptides with the help of UTY 246-254 -specific T-cell hybridomas in
lacZ assays.
(1) Remove spleen of one male and one female mouse and take up spleen in 5 ml
RPM!
10% FCS.
(2) Make a single-cell suspension by pressing spleen through a grid.
(3) Centrifuge cells for 5 min at 347 x g and discard supernatant.
(4) Lyse the erythrocytes by resuspending cells in 5 ml pre-warmed 1.66% (w/v)
NH 4CI
solution (in 15-ml tubes).
(5) Incubate for 2 min at room temperature.
(6) Fill up to 15 ml with RPMI 10% FCS and centrifuge cells for 5 min at 347 x
g and discard
supernatant.
(7) Wash cells with 15 ml PBS, centrifuge cells for 5 min at 347 x g, and
discard supernatant.
(8) Take up cells in 5 ml RPMI 10% FCS and count cells using a Neubauer
chamber.
(9) Incubate 10 7 splenocytes in 3 ml RPM! 10% FCS per well (6-well tissue
culture plate).
(10) Add desired amounts of inhibitor. You need one well of male splenocytes
without
inhibitor for comparison of untreated and treated samples. For female
splenocytes, you only
need one well without inhibitor.
(11) Incubate overnight at 37 C.
(12) Harvest splenocytes, wash cells twice with 15 ml PBS, and count
splenocytes.
(13) Resuspend cells in RPM! 10% FCS at 107/ml.
(14) Use 96-well round-bottom tissue culture plate and add 150 pl per well to
wells A1-D1.
Make four serial threefold dilutions of splenocytes (100 p1/per well).
(15) Harvest 1-cell hybridomas, count, and resuspend in RPM! 10% FCS at
10^6/ml. (We
use the UTY 246-254 -specific 1-cell hybridoma (5).)
(16) Add 100 pl of T-cell hybridomas per well (A1-A4; B1-134). Add to half of
your samples
(C1-C4; D1-D4) 100 pl RPM! 10% FCS as background control.
(17) Female splenocytes are used as negative control and untreated male
splenocytes as
positive control and for comparison. You can make an additional positive
control adding
synthetic peptide (we use UTY 246-254 peptide at a concentration of 10^-7 M)
to female
splenocytes.
(18) Incubate o/n at 37 C.
CA 03026564 2018-12-04
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PCT/EP2017/063699
(19) Centrifuge plate at 541 x g for 90 s and discard supernatant.
(20) Add 100 pl lacZ buffer and incubate at 37 C.
(21) Measure absorbance at 570/620 nm when colour change is visible (approx.
after 1-3 h).
Example 4
Alternative synthesis
Synthesis of tripeptide 28584
1 1 1
0 ,0 0 0 ro
o H o
boc,Nf NX1r r OH boc 0
H2Nfi "bn-u- boc, N bn K
H2NAIrN")1µ'0"bn
0
0 0 0 H
0 0
0 0
28579 28580 28581 I 28582 I
ii
A.Iprc%11`)% /Li
28684
28683
Scheme 1: Synthesis route of tripeptide 28584.
The synthesis of the tripeptide 28584 was successfully carried out according
to the literature
procedure. The formation of the Benzylester 28579 was performed in 4 g scale
and 4.6 g
product was obtained (>95% purity by 1H-NMR, 98% purity by LC/MS, 82% yield).
The following Boc-deprotection of 28579 led to 4.8 g of 28580 in quant. yield
and 95% purity
by 1H-NMR and 86% purity by LC/MS.
The peptide coupling was performed on a 2 g scale and 1.8 g of 28581 was
obtained (95%
purity by LC/MS, 90% purity by 1H-NMR, 73% yield).
Synthesis of 28582 was carried out in 1.7 g scale and 1.7 g product (28582)
was obtained as
TFA salt (quant. yield, 90% purity by 1H-NMR).
After amide formation of 2-Methyl-5-thiazolecarboxylic acid with 28582, 1.5 g
of 28583 was
obtained (80% yield, 93% purity by 1H-NMR; >95% by LC/MS).
,
CA 03026564 2018-12-04
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The benzyl ester deprotection of 28583 with 10% Pd/C was performed on 1.5 g
scale and 1.0
g 28584 was obtained (79% yield, 95% purity by 11-I-NMR and LC/MS). It was
found out that
the benzyl ester deprotection requires more than catalytic amount 10% Pd/C.
For fast
deprotection at least equal amount of Pd/C is needed compared to the used
amount of ,
28583.
.
,
Synthesis of NHS-Ketoester 28880 and thioester 29502, 29865
,
,
,
,
lic. OA N '74 ED:' MAP' - icoAg - ' X0IN -Niro 141
.
H 0 ID,i 0 99 H 1-1
lifteldn.cna acid 0 0
0.õ..,.
28864 /\ 28865
..., Mel, .
%-= ivZO,
E
-ir
P"'eptide N (:)." * HaN ')
' ) - - D-- ¨ X01
. 1109t, TFA, il
0 0 0 ar2CT2 0 0
28878 Pagide. 28077 2' 9f
,
I
o
o 0
F 1 F12. PEW
methanol Pt lit
peyll'Nfyy- OH
0
;.... 0
I
,rx0 Kfir pi Ar(ry,roli_
0
0 G US2S14/0113855A
."(sYrOH
PePhde N sA S 0 k.. C.,
H 144-lyeroxy- i.- 0 0
succi nmide I
28879 EGO 28880
= SN.Ac, DX, DMAP SNAc. DCC, DMAP
t
I
0 1.(N-y,,A.t4 s di
0 I
I
29502 29665
Scheme 2: Synthesis of NHS-Ketoester 28880 or thioester 29502 and 29865.
,
,
,
,
,
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39
PCT/EP2017/063699
The formation of the Meldrum's acid intermediate 28864 was performed by using
N-Boc-L-
Leu-OH and EDC/DMAP in DCM as reported in the literature. It was observed that
under
those reaction conditions product with complex mixture of side products was
formed. One
major side product was identified by LC/MS as cyclized Leucine derivative. The
reaction
crude has a purity of approx. 30-40% determined by 1H-NMR.
XolN EDC/DMAP XolN 0 X0j54
OH DCM
o o
===K cyclized side
product
28864
Scheme 3: Reaction of N-Boc-L-Leu-OH to 28864 and the formation of observed
cyclized
side product.
When 28864 was treated with benzyl alcohol for Meldrum's acid building block
opening the
desired product 28865 was obtained. From 6.7 g reaction 1.7 g of 28865 was
obtained (26%
yield, 85% purity by 11-I-NMR).
The double alkylation of 28865 was carried out by using excess of methyl
iodide in the
presence of K2CO3 in acetone which led to the formation of 28895 in 30-38%
yield. In the
end, out of a 3.7 g scale reaction 1.3 g of 28895 was obtained (38% yield, 95%
purity by 1H-
NMR).
The N-Boc deprotection of 28877 was performed using 10% TFA in DCM and was
done as
reported in the literature. After work up 1H-NMR clearly showed the mixture of
compounds.
Another reaction was carried out and it was found out that the reaction was
completed in two
hours instead of 17 h which was reported in the literature.
The reaction mixture was concentrated at room temperature.
The deprotection was performed shortly before the amide coupling of 28878. 400
mg
reaction of 28877 was carried out and the reaction crude (purity 85% by LC/MS)
was used
immediately for the next reaction step.
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PCT/EP2017/063699
HATU was used as a coupling reagent for the synthesis of 28878 successfully.
After work up =
the 11-I-NMR indicated that epimerization was occurred (15% by 1H-NMR). This
is
corresponding to the reported literature. The synthesis of 28878 was
successfully performed
in a 100 mg and 300 mg scale by using HATU as coupling reagent. After
purification 65 mg
(36% yield, purity 95% by LC/MS) and 298 mg (55% yield, purity 95% by LC/MS)
product
28878 was obtained. In both cases the presence of epimer was decreased from 15
mol% to
8 mol%.
The synthesis of 28879 was performed using N,N-dicyclohexylcarbodiimide and N-
hydroxysuccinimide in THF/DMF as solvent system. By LC/MS the formation of a
peak with
the right mass was observed.
After precipitation of dicyclohexylurea in DCM and removal of excess of NHS by
washing
with water, 25 mg were obtained (48% purity by LC/MS). The crude compound was
tried to
purify by preparative HPLC on reverse phase column. The fractions were
extracted by DCM
in order to avoid hydrolysis/decarboxylation. 0.7 mg with 93% purity and a
peak with the right
product mass in LC/MS was obtained. 1H-NMR in CDCI3 provided no clear
indication due to
the small amount of sample.
For structure elucidation, reaction of 28880 was performed on a 140 mg scale
by using N,N-
dicyclohexylcarbodiimide and N-hydroxysuccinimide (NHS) in Et0Ac/DMF as
solvent
system. LC/MS analysis showed the formation of a peak with the right product
mass. After
precipitation of dicyclohexylurea in DCM and removal of excess of NHS by
washing with
water, 120 mg were obtained (48% purity by LC/MS). The crude was tried to
purify by
preparative HPLC.
To enhance the coupling efficiency PyBOP was used as a coupling reagent. By
using 1.3 eq.
of PyBOP, 2.0 eq. DIPEA and 3.0 eq. NHS a very prominent peak with right
product mass
was observed also in much higher intensity than before.
In order to prove whether the peak with the right product mass observed in
LC/MS is an
artefact or belongs to the desired compound 28880, it was treated with Me0H to
form the
methylester of 28880. LC/MS analysis showed that the peak with the mass of the
expected
NHS-ester was disappeared and the formation of a peak with the right mass of
the
methylester was observed.
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PCT/EP2017/063699
Purification was done by doing two times short plug filter column
chromatography. In the first
case the reaction mixture was directly filtered through a pad of silica
without any solvent
removal after the reaction. THF was used as eluent. After concentration of
this above
mentioned fraction, compound was purified again by short column chromatography
using
.. DCM/THF mixture as eluent. 9.8 mg of product was obtained (62% purity by
LC/MS). Due to
very small amount the purity of product cannot be improved further. However,
11-I-NMR is
corresponding to the product. For larger scale the purification was improved
by optimizing
the column chromatography using c-hexane/THF as solvent mixture.
Another 100 mg reaction was performed by using 1.3 eq. of PyBOP, 2.0 eq. DIPEA
and 3.0
eq. NHS as coupling conditions. Purification was done by doing short plug
filter column
chromatography. In the first case the reaction mixture was filtered through a
pad of silica
without any concentration. THF was used as eluent. After concentration of the
above
mentioned fraction, compound was purified by column chromatography using c-
hexane/THF
mixture as eluent. 47 mg of desired title compound 28880 was obtained with
purity of 88% by
1H-NMR, containing 13.5 mol% of decarboxylated 28879.
In conclusion, 47 mg of 28880 was delivered successfully in 88% purity by 1H-
NMR.
