U.S. patent application number 17/567280 was filed with the patent office on 2022-04-28 for small molecules.
The applicant listed for this patent is University of Dundee. Invention is credited to Alessio Ciulli, Scott J. Hughes, Chiara Maniaci, Andrea Testa.
Application Number | 20220127257 17/567280 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-28 |
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United States Patent
Application |
20220127257 |
Kind Code |
A1 |
Ciulli; Alessio ; et
al. |
April 28, 2022 |
SMALL MOLECULES
Abstract
Compounds having the general structure A-L-B are presented
wherein A and B are independently an E3 ubiquitin ligase protein
binding ligand compound of formula 1A or 1B. Pharmaceutical
compositions comprising these compounds and methods of use are also
presented.
Inventors: |
Ciulli; Alessio; (Dundee,
GB) ; Maniaci; Chiara; (Dundee, GB) ; Hughes;
Scott J.; (Dundee, GB) ; Testa; Andrea;
(Dundee, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Dundee |
Dundee |
|
GB |
|
|
Appl. No.: |
17/567280 |
Filed: |
January 3, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16604737 |
Oct 11, 2019 |
11261179 |
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PCT/GB2018/050987 |
Apr 13, 2018 |
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17567280 |
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International
Class: |
C07D 417/14 20060101
C07D417/14 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2017 |
GB |
1706042.7 |
Apr 14, 2017 |
GB |
1706043.5 |
Claims
1. A compound having the structure: A-L-B wherein A and B are
independently an E3 ubiquitin ligase protein binding ligand
compound of formula 1A or 1B and at least one of A or B is the
compound of formula 1B: ##STR00056## wherein L is a linking group
which is directly bonded to the compound of formula 1A at R.sup.1
or R.sup.2, and/or directly bonded to the compound of formula 1B at
R.sup.3 or R.sup.4 and wherein L is
--R.sup.5--[O(CH.sub.2).sub.m].sub.n--R.sup.6--, wherein m and n
are independently 0 to 10, and R.sup.5 and R.sup.6 are
independently selected from the group: covalent bond, C1-C10
alkylene, --OR.sup.7--, C1-C10 polyether, or --O--; wherein R.sup.1
is selected from either the group: (1) a covalent bond, or C1-C5
alkylene when L is bonded to the compound of formula 1A at R.sup.1,
or the group (2) H, NH.sub.2, C1-C5 alkyl, or C(CN)C.sub.2H.sub.4
when L is bonded to the compound of formula 1A at R.sup.2; wherein
R.sup.2, R.sup.3, and R.sup.4 are independently selected from the
group: a covalent bond, H, NH.sub.2, C1-C5 alkyl,
C(CN)C.sub.2H.sub.4; wherein X and Y are independently selected
from the group: H, OH or halogen; and wherein R.sup.7 is C1-C5
alkylene, or a pharmaceutically acceptable salt, hydrate, solvate
or polymorph thereof.
2. A compound according to claim 1, wherein X is H or halogen.
3. A compound according to claim 1, wherein Y is OH.
4. A compound according to claim 1, wherein either A or B is a
compound according to formula 1A, wherein A has the formula 1C:
##STR00057##
5. A compound according to claim 1, wherein A is a compound of
formula 1A and B is a compound of formula 1B.
6. A compound according to claim 1, wherein L is connected to A via
R.sup.1 of formula 1A.
7. A compound according to claim 1, wherein L is connected to B via
R.sup.1 of formula 1A.
8. A compound according to claim 1, wherein R.sup.5 is a chemical
bond, R.sup.6 is a chemical bond, m is 2 and n is 3, 4 or 5.
9. A compound according to claim 8, wherein n is 5.
10. A compound according to claim 1, wherein the linker L is a
linear chain of 12-20 atoms in length.
11. A compound according to claim 10, wherein the linker chain
comprises carbon and/or oxygen atoms.
12. A compound according to claim 11, wherein the linker chain
comprises alkylene groups and/or ether groups and/or polyether
groups.
13. A pharmaceutical composition comprising one or more compounds
according to claim 1 and a pharmaceutically acceptable vehicle or
diluent therefor.
14. A method of use of a compound according to claim 1 for the
treatment of at least one of anaemia due to chronic kidney disease,
anaemia due to cancer chemotherapy, ischemia, ischemic reperfusion
injuries, myocardial infarction, stroke, acute lung injury,
intestinal inflammation, wound healing and post-transplantation
complications, mitochondrial respiratory chain dysfunctions and
oncological conditions treatable by enhancing T-cell responses.
15. A method of regulating activity of a target protein in a
subject comprising administering to said subject an effective
amount of a compound according to claim 1.
16. The method according to claim 15, wherein the target protein is
an E3 ubiquitin ligase protein.
Description
FIELD OF THE INVENTION
[0001] This invention relates to small molecule E3 ubiquitin ligase
protein binding ligand compounds, and to their utility in
PROteolysis Targeted Chimeras (PROTACs), as well as processes for
the preparation thereof, and use in medicine. This invention
particularly relates to PROTACs capable of inducing
auto-ubiquitination of E3 ubiquitin ligases and triggering their
subsequent proteasomal degradation.
BACKGROUND OF THE INVENTION
[0002] E3 ubiquitin ligases are emerging as attractive targets for
small-molecule modulation and drug discovery. E3s bring a substrate
protein and ubiquitin in close proximity to each other to catalyze
the transfer of a ubiquitin molecule to the substrate. Substrate
ubiquitination can trigger different cellular outcomes, of which
one of the best characterized is poly-ubiquitination and subsequent
proteasomal degradation. The human genome comprises >600
predicted E3 ligases that play important roles in normal cellular
physiology and disease states, making them attractive targets for
inhibitor discovery. However, E3 ligases do not comprise deep and
"druggable" active sites for binding to small molecules. Blockade
of E3 ligase activity therefore requires targeting of
protein-protein interactions (PPIs), and the often extended, flat
and solvent-exposed PPI surfaces make it a challenge for drug
design. Only a few potent inhibitors have been developed to date,
mostly compounds that bind to the E3 substrate recognition site,
for example MDM2, inhibitor of apoptosis proteins (IAPs), the von
Hippel-Lindau (VHL) ligase,.sup.1-3 and KEAP1. Inhibitors of
E3:substrate interaction can exhibit a discrepancy in effective
concentrations between biophysical binding and cellular
efficacy,.sup.3 due to competition from high-affinity endogenous
substrates that markedly increase their cellular concentration as a
consequence of the inhibition. This poses limitations, such as the
need to use high inhibitor concentrations, which can lead to
off-target effects and cytotoxicity, and incomplete blockade of
enzyme activity. Moreover, E3 ligases are multi-domain and
multi-subunit enzymes, and targeting an individual binding site
leaves other scaffold scaffolding regions untouched and other
interactions functional. As a result, E3 ligase inhibition may be
ineffective or fail to recapitulate genetic knockout or knockdown.
New chemical modalities to target E3 ligases are therefore
demanded.
[0003] E3 ligases are not merely targets for inhibition. Compounds
of natural or synthetic origin have been discovered that bind to E3
ligases and promote the recruitment of new proteins. These
interfacial compounds induce de novo formation of ligase-target
PPIs effectively hijacking E3 ubiquitination activity towards the
neo-substrates, for targeted protein degradation. One class of
small molecule hijackers of E3 ligase activity comprises monovalent
compounds. These so-called "molecular glues" include the plant
hormone auxin, which binds to the Cullin RING ligase (CRL)
CRL1-TIR1 to target transcriptional repressor proteins of the
Aux/IAA family, and the immunomodulatory drugs (IMiDs) thalidomide,
lenalidomide, pomalidomide and analogue CC-885, that all bind to
cereblon (CRBN), a subunit of the CRL4-CRBN ligase, and redirect
CRBN activity to different substrates..sup.4-10 More recently, the
sulfonamide anti-cancer drug indisulam was found to induce
degradation of the splicing factor RBM39 via recruiting CRL4-DCAF15
activity. A distinct class of compounds that display a similar
mechanism of action are bivalent molecules called
Proteolysis-Targeting Chimeras (PROTACs). PROTACs comprise of a
first warhead moiety for a ligase, and a second warhead for a
target protein, joined by a linker..sup.11 Formation of a ternary
complex between the PROTAC, the ligase and the target triggers
proximity-induced target ubiquitination and degradation. Warhead
ligands have been used to develop potent and cell-active PROTACs
recruiting different ligases, including CRL2-VHL,.sup.12-15
CRL4-CRBN,.sup.16-20 and IAPs..sup.21-22 Amongst the targets
successfully degraded by PROTACs are BET proteins Brd2, Brd3 and
Brd4,.sup.12,14-17 FKBP,.sup.16,20 protein kinases,.sup.13,18
amongst others.sup.13,21 An attractive feature of PROTACs is their
sub-stoichiometric catalytic activity,.sup.13 which does not
require full occupancy of the target-binding site as with
conventional inhibitors, leading to degrading concentrations that
can be orders of magnitude lower than the inhibitory concentrations
of their constitutive parts alone. Furthermore, induced target
depletion can have a more sustained cellular effect compared to
target inhibition, and can overcome compensatory cellular feedback
mechanisms, such as increase in target levels. Crucially, it has
been shown that PROTAC molecules can exhibit an added layer of
selectivity for protein degradation beyond the intrinsic binding
selectivity of the warhead ligand.sup.12,15,18 Our recent
structural work with Brd4-selective PROTACs targeting CRL2-VHL
revealed that the importance of specific ligand-induced PPIs
between the ligase and the target, which contribute to cooperative
formation of stable and highly populated ternary
complexes..sup.15
[0004] The inventors have now found that it is possible to target
E3 ligases themselves for ubiquitination and proteasomal
degradation, using a suitably designed PROTAC. For at least some
aspects the inventors have found that a PROTAC comprising two
instances of an E3 binding moiety may be capable of forming ternary
complexes in which the same E3 functions as both ubiquitinating
enzyme and neo-substrate.
SUMMARY OF THE INVENTION
[0005] According to a first aspect of the invention there is
provided a compound having the structure:
A-L-B
[0006] wherein A and B are independently an E3 ubiquitin ligase
protein binding ligand compound of formula 1A or 1B:
##STR00001##
[0007] wherein L is a linking group which is directly bonded to the
compound of formula 1A at R.sup.1 or R.sup.2, and/or directly
bonded to the compound of formula 1B at R.sup.3 or R.sup.4 and
wherein L is --R.sup.5--[O(CH.sub.2).sub.m].sub.n--R.sup.6--,
wherein m and n are independently 0 to 10, and R.sup.5 and R.sup.6
are independently selected from the group: covalent bond, C1-C10
alkylene, C1-C10 polyether, or --O--;
[0008] wherein R.sup.1 is selected from either the group: (1) a
covalent bond, or C1-C5 alkylene when L is bonded to the compound
of formula 1A at R.sup.1, or the group (2) H, NH.sub.2, C1-C5
alkyl, or C(CN)CH.sub.4when L is bonded to the compound of formula
1A at R.sup.2;
[0009] wherein R.sup.2, R.sup.3, and R.sup.4 are independently
selected from the group: a covalent bond, H, NH.sub.2, C1-C5 alkyl,
C(CN)C.sub.2H.sub.4;
[0010] wherein X and Y are independently selected from the group:
H, OH or halogen; and
[0011] wherein R.sup.7 is C1-C5 alkylene,
[0012] or a pharmaceutically acceptable salt, hydrate, solvate or
polymorph thereof.
[0013] Accordingly, the compound of formula A-L-B may comprise a
compound of either formula 1A or 1B connected via the linker L to a
compound of either formula 1A or 1B. In embodiments where A and/or
B is a compound of formula 1A, the compound of formula 1A may be
connected to the linker L via R.sup.1 or R.sup.2. In embodiments
where A and/or B is a compound of formula 1B, the compound of
formula 1B may be connected to the linker L via R.sup.3 or
R.sup.4.
[0014] Compounds having the general formula A-L-B as described
herein may be referred to in the description below as
"PROTAC-compounds", "HOMO-PROTAC compounds" (wherein the moiety A
is the same as the moiety B), "Hetero-PROTAC compounds" (wherein
the moiety A is different to moiety B), or simply as "compounds of
the invention".
[0015] The inventors have surprising found that the compounds
having the structure A-L-B as defined above are able to induce
degradation of E3 ubiquitin ligase protein within a cell by using
the E3 ubiquitination mechanism itself. Accordingly, it suggested
that the compounds of structure A-L-B forms a tertiary structure
with two E3 ubiquitin ligase proteins such that one E3 ubiquitin
ligase protein ubiquitinates another E3 ubiquitin ligase protein to
which it is joined by the compound of structure A-L-B. It is
further suggested that this ubiquitination is induced due to the
enforced close proximity of the two E3 ubiquitin ligase proteins in
the tertiary structure formed by binding of the E3 ubiquitin ligase
proteins with the compounds of formula 1A or 1B.
[0016] Furthermore, it has been found that the compounds of the
invention are able to initiate the degradation at
sub-stoichiometric concentrations, thereby indicating that the
compounds are at least partially catalysing the degradation.
[0017] In some embodiments X may be H or halogen.
[0018] In embodiments where X is a halogen, X may be selected from
F, Cl, Br, or I. For example, X may be selected from F or Cl. X may
be F.
[0019] In some embodiments, Y may be OH. Typically, Y is in the
"down" position as illustrated in formula 1C below.
[0020] In embodiments where either A or B is a compound according
to formula 1A, A or B may have the formula 1C:
##STR00002##
[0021] In some embodiments, A may be a compound of formula 1A and B
may be a compound of formula 1A.
[0022] L may be connected to A via R.sup.1 of formula 1A. L may be
connected to B via R.sup.1 of formula 1A.
[0023] Alternatively, L may be connected to A via R.sup.2 of
formula 1A and L may be connected
[0024] In some embodiments, R.sup.5 may be a chemical bond, R.sup.6
may be a chemical bond, m may be 2 and n may be 3, 4 or 5.
[0025] In some preferred embodiments, n is 5.
[0026] The compound of some embodiments may have formula 2, 3 or
4:
##STR00003##
[0027] wherein R.sup.2a, R.sup.2b and R.sup.2c are independently
selected from H, NH.sub.2, C1-C5 alkyl, and
C(CN)C.sub.2H.sub.4;
[0028] R.sup.1a, R.sup.1b and R.sup.1c are independently selected
from H, NH.sub.2, C1-C5 alkyl, and C(CN)C.sub.2H.sub.4;
[0029] X.sup.1 and X.sup.2 are independently selected from H, OH,
halogen;
[0030] Y.sup.1 and Y.sup.2 are independently selected from H, OH,
halogen; and
[0031] m and n are independently 0 to 10.
[0032] Preferably for compounds of formula 2, 3 or 4, n is 3-5.
Typically, m is 1-4. Preferably, m is 2 such that the linker is
formed of polyethylene glycol subunits.
[0033] In embodiments, R.sup.1a, R.sup.1b and R.sup.1c may be
independently selected from C1-C5 alkyl or C(CN)C.sub.2H.sub.4. In
further embodiments, R.sup.1a, R.sup.1b and R.sup.1c may be
independently selected from C1 alkyl (i.e. methyl or Me) and
C(CN)C.sub.2H.sub.4.
[0034] In some embodiments R.sup.2a, R.sup.2b and R.sup.2c may be
H.
[0035] In some embodiments, Y.sup.1 and Y.sup.2 may be OH, X.sup.1
and X.sup.2 may be H, R.sup.1a, R.sup.1b and R.sup.1c may
independently be Me or C(CN)C.sub.2H.sub.4 and R.sup.2a, R.sup.2b
and R.sup.2c may be H.
[0036] In preferred embodiments the linker L is a linear chain of
12-20 atoms in length. The compounds of the invention have been
found to be most useful to induce degradation of target proteins
when the groups A and B are spaced apart. Accordingly, without
wishing to be bound by theory, it has been found that a linker L
being a linear chain of 12-20 atoms in length spaces the groups A
and B apart a sufficient distance to allow them to bind to their
target binding sites without interfering with one another, whilst
at the same time ensuring that the target proteins are held in
sufficient proximity that the E3 ubiquitin ligase protein bound to
either or both A and B can ubiquitinate the target protein, thereby
marking that protein for subsequent degradation by the cell's
machinery.
[0037] L may be a linear chain of 15-18 atoms in length. For
example, L may be a linear chain of 15, 16, 17 or 18 atoms in
length.
[0038] Typically, the linker chain may comprise carbon and/or
oxygen atoms. For example, the linker chain may comprise alkylene
groups and/or ether groups and/or polyether groups.
[0039] Alternatively, the linker chain may be a peptide chain, or
nucleotide chain, for example.
[0040] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts of the compounds formed by the process of the
present invention which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable
benefit/risk ratio. Pharmaceutically acceptable salts are well
known in the art. The salts can be prepared in situ during the
final isolation and purification of the compounds of the invention,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable salts
suitable for use herein include, but are not limited to, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, maleic acid, tartaric acid, citric acid,
succinic acid or malonic acid or by using other methods used in the
art such as ion exchange.
[0041] Other pharmaceutically acceptable salts include, but are not
limited to, adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carbon/late, sulfate,
phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,
sulfonate and aryl sulphonate.
[0042] In a preferred aspect herein the compounds of formula I for
use in the PROTAC compounds of structure A-L-B- as defined herein
are represented as a defined stereoisomer. The absolute
configuration of such compounds can be determined using art-known
methods such as, for example, X-ray diffraction or NMR and/or
implication from starting materials of known stereochemistry.
[0043] Pharmaceutical compositions in accordance with the invention
will preferably comprise substantially stereoisomerically pure
preparations of the indicated stereoisomer.
[0044] Pure stereoisomeric forms of the compounds and intermediates
as mentioned herein are defined as isomers which are substantially
free of other enantiomeric or diastereomeric forms of the same
basic molecular structure of said compounds or intermediates. In
particular, the term "stereoisomerically pure" concerns compounds
or intermediates having a stereoisomeric excess of at least 80%
(i.e. minimum 90% of one isomer and maximum 10% of the other
possible isomers) up to a stereoisomeric excess of 100% (i.e. 100%
of one isomer and none of the other), more in particular, compounds
or intermediates having a stereoisomeric excess of 90% up to 100%,
even more in particular having a stereoisomeric excess of 94% up to
100% and most in particular having a stereoisomeric excess of 97%
up to 100%. The terms "enantiomerically pure" and
"diastereomerically pure" should be understood in a similar way,
but then having regard to the enantiomeric excess, and the
diastereomeric excess, respectively, of the mixture in
question.
[0045] Pure stereoisomeric forms of the compounds and intermediates
as detailed herein may be obtained by the application of art-known
procedures. For instance, enantiomers may be separated from each
other by the selective crystallization of their diastereomeric
salts with optically active acids or bases. Examples thereof are
tartaric acid, dibenzoyl tartaric acid, ditoluoyltartaric acid and
camphorsulfonic acid. Alternatively, enantiomers may be separated
by chromatographic techniques using chiral stationary phases. Said
pure stereochemically isomeric forms may also be derived from the
corresponding pure stereochemically isomeric forms of the
appropriate starting materials, provided that the reaction occurs
stereo-specifically. Preferably, if a specific stereoisomer is
desired, said compound is synthesized by stereospecific methods of
preparation. These methods will advantageously employ
enantiomerically pure starting materials.
[0046] The diastereomeric racemates of the compounds of formula 1A
or 1B for use in the PROTAC compounds of structure A-L-B as defined
herein can be obtained separately by conventional methods.
Appropriate physical separation methods that may advantageously be
employed are, for example, selective crystallization and
chromatography, e.g. column chromatography.
[0047] According to a second aspect of the invention there is
provided a compound selected from the following group:
##STR00004##
[0048] In a preferred embodiment, the compound is selected from the
group of compounds (7) to (13). For example, the compound may be
compound (7).
[0049] The invention extends in a third aspect to a pharmaceutical
composition comprising one or more compounds according to the first
or second aspect and a pharmaceutically acceptable vehicle or
diluent therefor.
[0050] PROTAC compounds of the invention can be administered as
pharmaceutical compositions by any conventional route, in
particular enterally, e.g., orally, e.g., in the form of tablets or
capsules, or parenterally, e.g., in the form of injectable
solutions or suspensions, topically, e.g., in the form of lotions,
gels, ointments or creams, or in a nasal or suppository form.
Pharmaceutical compositions comprising a PROTAC compound of the
present invention in free form or in a pharmaceutically acceptable
salt form in association with at least one pharmaceutically
acceptable carrier or diluent can be manufactured in a conventional
manner by mixing, granulating or coating methods. For example, oral
compositions can be tablets or gelatin capsules comprising the
active ingredient together with a) diluents, e.g., lactose,
dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)
lubricants, e.g., silica, talcum, stearic acid, its magnesium or
calcium salt and/or polyethyleneglycol; for tablets also c)
binders, e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth, methylcellulose, sodium carboxymethylcellulose and or
polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches,
agar, alginic acid or its sodium salt, or effervescent mixtures;
and/or e) absorbents, colorants, flavors and sweeteners. Injectable
compositions can be aqueous isotonic solutions or suspensions, and
suppositories can be prepared from fatty emulsions or suspensions.
The compositions may be sterilized and/or contain adjuvants, such
as preserving, stabilizing, wetting or emulsifying agents, solution
promoters, salts for regulating the osmotic pressure and/or
buffers. In addition, they may also contain other therapeutically
valuable substances. Suitable formulations for transdermal
applications include an effective amount of a PROTAC compound of
the present invention with a carrier. A carrier can include
absorbable pharmacologically acceptable solvents to assist passage
through the skin of the host. For example, transdermal devices are
in the form of a bandage comprising a backing member, a reservoir
containing the compound optionally with carriers, optionally a rate
controlling barrier to deliver the compound to the skin of the host
at a controlled and predetermined rate over a prolonged period of
time, and means to secure the device to the skin. Matrix
transdermal formulations may also be used. Suitable formulations
for topical application, e.g., to the skin and eyes, are preferably
aqueous solutions, ointments, creams or gels well-known in the art.
Such may contain solubilizers, stabilizers, tonicity enhancing
agents, buffers and preservatives.
[0051] The pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of a PROTAC compound of
the present invention formulated together with one or more
pharmaceutically acceptable carriers. As used herein, the term
"pharmaceutically acceptable carrier" means a non-toxic, inert
solid, semi-solid or liquid filler, diluent, encapsulating material
or formulation auxiliary of any type.
[0052] The pharmaceutical compositions of this invention can be
administered to humans and other animals orally, rectally,
parenterally, intracisternally, intravaginally, intraperitoneally,
topically (as by powders, ointments, or drops), buccally, or as an
oral or nasal spray.
[0053] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
compounds, the liquid dosage forms may contain inert diluents
commonly used in the art such as, for example, water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring,
and perfuming agents.
[0054] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides.
[0055] In addition, fatty acids such as oleic acid are used in the
preparation of injectables. In order to prolong the effect of a
drug, it is often desirable to slow the absorption of the drug from
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material with poor water solubility. The rate of absorption of the
drug then depends upon its rate of dissolution which, in turn, may
depend upon crystal size and crystalline form. Alternatively,
delayed absorption of a parenterally administered drug form is
accomplished by dissolving or suspending the drug in an oil
vehicle. Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the PROTAC
compounds of the invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound. Solid compositions of a
similar type may also be employed as fillers in soft and
hard-filled gelatine capsules using such excipients as lactose or
milk sugar as well as high molecular weight polyethylene glycols
and the like.
[0056] The PROTAC compounds can also be provided in
micro-encapsulated form with one or more excipients as noted above.
The solid dosage forms of tablets, dragees, capsules, pills, and
granules can be prepared with coatings and shells such as enteric
coatings, release controlling coatings and other coatings well
known in the pharmaceutical formulating art. In such solid dosage
forms the active compound may be admixed with at least one inert
diluent such as sucrose, lactose or starch. Such dosage forms may
also comprise, as is normal practice, additional substances other
than inert diluents, e.g., tableting lubricants and other tableting
aids such a magnesium stearate and microcrystalline cellulose. In
the case of capsules, tablets and pills, the dosage forms may also
comprise buffering agents.
[0057] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, ear drops, eye
ointments, powders and solutions are also contemplated as being
within the scope of this invention. The ointments, pastes, creams
and gels may contain, in addition to an active compound of this
invention, excipients such as animal and vegetable fats, oils,
waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof.
[0058] Powders and sprays can contain, in addition to the PROTAC
compounds of this invention, excipients such as lactose, talc,
silicic acid, aluminium hydroxide, calcium silicates and polyamide
powder, or mixtures of these substances. Sprays can additionally
contain customary propellants such as chlorofluorohydrocarbons.
Transdermal patches have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0059] In a fourth aspect, the invention provides a PROTAC compound
of structure A-L-B as defined herein for use as a medicament.
[0060] In a fifth aspect of the invention there is provided a
method of use of a compound according to any of the first or second
aspect or a pharmaceutical composition according to the third
aspect for the treatment of at least one of anaemia due to chronic
kidney disease.sup.23, anaemia due to cancer chemotherapy.sup.24,
ischemia.sup.25, ischemic reperfusion injuries.sup.26, myocardial
infarction.sup.27, stroke.sup.27, acute lung injury.sup.28,
intestinal inflammation.sup.29, wound healing.sup.30 and
post-transplantation complications.sup.31, mitochondrial
respiratory chain dysfunctions.sup.32 and oncological conditions
treatable by enhancing T-cell responses.sup.33.
[0061] According to a sixth aspect of the invention there is
provided a method of regulating activity of a target protein in a
subject comprising administering to said subject a therapeutically
effective amount of a compound according to the first or second
aspect, or a pharmaceutical composition according to the third
aspect.
[0062] The term "subject" as used herein refers to a mammal. A
subject therefore refers to, for example, dogs, cats, horses, cows,
pigs, guinea pigs, and the like. Preferably the subject is a human.
When the subject is a human, the subject may also be referred to
herein as a patient.
[0063] The term "therapeutically effective amount" means an amount
effective to treat, cure or ameliorate a disease, condition illness
or sickness.
[0064] Preferably, the target protein is an E3 ubiquitin ligase
protein. Typically the E3 ubiquitin ligase protein is selected from
CRL2-VHL, CRL4-CRBN. The E3 ubiquitin ligase protein may be
selected from any of the >230 cullin RING ligases, for example
CRL1-Skp2, CRL1-bTrCP, CRL1-Fbw, CRL1-Fbxo, CRL1-Fbxl, CRL2-LRR1,
CRL2-FEM1, CRL3-Keap1, CRL3-KLHL, CRL3-SPOP, CRL4-DDB2, CRL4-DCAF,
CRL4-CSA, CRL4-CDT2, CRL5-SOCS, CRL5-ASB. Other E3 ubiquitin ligase
proteins may be selected from MDM2, c-Cbl, APC-C, FANCL, UBE3A,
UBE3B, UBE3C, UBE3D, Parkin, SIAH, XIAP, UHRF1, TRAF6, PELI2, RNF2,
RNF4 amongst others.
[0065] Preferred and optional features of the first to sixth
aspects may be preferred and optional features of the other of the
first to sixth aspects as appropriate.
BRIEF DESCRIPTION OF THE FIGURES
[0066] Embodiments of the present invention will now be described,
by way of non-limiting example, with reference to the accompanying
drawings.
[0067] FIG. 1: (a) Crystal structures of VHL in complex with VH298
(PDB code 5LLI). VHL is shown in surface representation and the
bound ligand as sticks representation. (b) Chemical structure of
VHL inhibitors VH032 and VH298.
[0068] FIG. 2: General chemical structure and design of
Homo-PROTACs compounds. Linkage sites at acetyl and phenyl group
are indicated.
[0069] FIG. 3: Synthesis of Homo-PROTACs compounds symmetric from
acetyl group CM09, CM10, CM11 and negative control compound
CMP98.
[0070] FIG. 4: Synthesis of negative control Homo-PROTAC compound
CMP99 with cis-trans configuration.
[0071] FIG. 5: Synthesis of VHL binding moieties 17 and 18
[0072] FIG. 6: Synthesis of Homo-PROTACs CMP106 and CMP108
symmetrically derivatized from the phenyl group.
[0073] FIG. 7: Synthesis of asymmetric Homo-PROTACs CMP112 and
CMP113.
[0074] FIG. 8: Biological evaluation of HOMO-PROTACs. (a) HeLa
cells were treated with 0.1% DMSO, VH032 (150 .mu.M) and 1 .mu.M of
the indicated compounds for 10 h. Abundance of individual proteins
was analyzed by Western blotting using corresponding specific
antibodies accordingly after SDS-PAGE. (b) Different cells lines
were treated with si-RNA targeting VHL proteins or negative control
si-RNA (for 48 h), as well as with CM11 (1 .mu.M) or 0.1% v/v DMSO
for 10 h.
[0075] FIG. 9: HeLa cells were treated with increasing
concentration of HOMO-PROTAC CM11 for 4 h or 24 h.
[0076] FIG. 10: Time-course immunoblots of lysates from HeLa cells
subjected to 0.1% DMSO, CoCl2 (100 mM), IOX2 (150 mM), VH032 (250
mM or 1 mM) or 1 mM of CM11.
[0077] FIG. 11. Compound activity is CRL2VHL and proteasome
dependent. HeLa cells treated with CM11 in the absence or presence
of proteasome inhibitor MG132, MLN4924, VHL inhibitor VH032 or PHD2
inhibitor IOX4.
[0078] FIG. 12. Biophysical studies of Homo-PROTACs binding to VHL.
(a) Superposition of the integrated ITC heat curves of CM11 CMP99
or CMP98 titrations against VCB. (b) SEC assay of complex formation
after incubation of CM11, CMP98, CMP99, VH032 or DMSO with VCB. (c)
AlphaLISA: intensity values titrating CM09, CM10, CM11 and CMP98
against VCB. Each point is mean (.+-.SEM) intensity of four
technical replicates.
[0079] FIG. 13: Proposed model for the mechanism of action of
Homo-PROTAC CM11.
[0080] FIG. 14: HeLa or U2OS cells stably expressing HRE-luciferase
reporter plasmid were treated with the indicated compounds at the
indicated concentrations for the indicated time.
[0081] FIG. 15: Dose-response curve of CA9 mRNA expression in HeLa
(16 h)
[0082] FIG. 16: Hela cells were treated with increasing
concentration of indicated compound for 4 h or 24 h.
[0083] FIG. 17: Concentration dependency experiment in U2OS (10 h
treatment)(left) and Time course experiments of lysate from U2OS
(right).
[0084] FIG. 18: Time-course immunoblots of lysates from HeLa cells
subjected to 0.1% DMSO, CoCl2 (100 .mu.M), IOX2 (150 .mu.M), VH032
(250 .mu.M or 1 .mu.M) or 1 .mu.M of indicated compounds.
[0085] FIG. 19: Integrated ITC heat curves of CM09 (a), CM10 (b),
and CM11 (c) against VCB.
[0086] FIG. 20: Superposition of the integrated ITC heat curves of
CM11, CM09, or CM10 titrations against VCB.
[0087] FIG. 21: SEC assay of complex formation after incubation of
CM11, CM09, CM10 or DMSO (black) with VCB.
[0088] FIG. 22: Immunomodulatory drugs targeting cereblon. (a)
Chemical structures. (b) Crystal structure of pomalidomide bound to
CRBN (PDB code 4Cl3).sup.5
[0089] FIG. 23: Structure of Hetero-PROTACs designed to recruit
CRL4.sup.CRBN at one end and CRL2.sup.VHL at the other end.
[0090] FIG. 24: Synthesis of intermediates 29 and 45.
[0091] FIG. 25: Synthesis of 52 (CMP85) and 51 (CMP86)
[0092] FIG. 26: Side product 53 of cyclization reaction.
[0093] FIG. 27: Chemical structures of CM09, CM10, CM11,
[0094] FIG. 28: HeLa, Hek293 and U2OS cells were treated with 1
.mu.M of CM09, CM10, CM11, DAT265, CMP85 or CMP86, 0.1% DMSO,
CoCl.sub.2 (100 .mu.M), IOX2 (50 .mu.M), IOX4 (50 .mu.M)
[0095] FIG. 29: Integrated ITC heat curve for CMP106 against
VCB.
[0096] FIG. 30: Integrated ITC heat curve for CMP108 against
VCB.
[0097] FIG. 31 Integrated ITC heat curve for CMP112 against
VCB.
[0098] FIG. 32: Integrated ITC heat curve for CMP113 against
VCB.
[0099] FIG. 33: Superposition of the integrated ITC heat curves for
CM09, CM10, CM11, CMP112, CMP113, CMP106, CMP98 and CMP99 against
VCB
DETAILED DESCRIPTION
[0100] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0101] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0102] Biology
[0103] Human cell lines HeLa, U2OS and HEK 293, purchased from
ATCC, were propagated in DMEM supplemented with 10% fetal bovine
serum (FBS), L-glutamine, 100 .mu.g ml.sup.-1 of
penicillin/streptomycin at 37.degree. C. and 5% CO.sub.2. Cells
were maintained for no more than 30 passages. All cell lines were
routinely tested for mycoplasma contamination using MycoAlert kit
from Lonza.
Small Interfering RNA.
[0104] For siRNA inhibition studies, 3.times.10.sup.5 cells were
seeded into each well of a 6-well plate in order to achieve 70% of
confluence on the day of transfection. siRNA (SMARTpool:
ON-TARGETplus VHL siRNA L-003936-00-0005) was prepared as a 20
.mu.M solution in RNase-free 1.times. siRNA buffer. Negative
control siRNA (siRNA from Life Technologies, cat. #4390843) was
used as negative control. On the day of transfection, old medium
was replaced with fresh one. siRNA solution (5 .mu.L) of both VHL
targeting siRNA and negative control were added to 250 .mu.L of
Opti-mem in 1.5 mL tube. This solution was prepared in duplicate.
The content in each tube was mixed by pipetting. Lipofectamine
RNAiMax (5 .mu.L) was added to 250 .mu.L of
[0105] Opti-mem in another 1.5 mL tube. The solution was prepared
in duplicate. The content in each tube was mixed by pipetting. The
solution from step 2 was added to the tube in step 3. The solution
was mixed by brief vortex ad incubated at r.t. for 20 min. The
tubes were centrifuged briefly. The whole volume of transfection
mix was added to the 6-well plate. Plate was swirled gently back
and forth to mix the content. Plates were incubated at 37.degree.
C. and 5% CO.sub.2 for 48 h before harvesting.
[0106] Single Point Treatment.
[0107] For single time point treatment experiments, cells were
transferred in 6-well plates with 5.times.10.sup.5 cells per well
in 2 ml media in order to achieve 80% confluence the following day.
Stock concentrations of compounds were prepared by solubilizing the
powder in 100% v/v DMSO to the final desired stock
concentration.
[0108] On the day of treatment, all compound samples were prepared
as 100-fold concentrated compound solution using DMEM just before
treatment. The experiment samples (20 .mu.L) were added to the 6
well plate containing 2 ml of media. The final DMSO concentration
was 0.1% v/v. Cells were incubated at 37.degree. C. and 5% CO.sub.2
for the desired time before harvesting.
[0109] Time Course Experiments.
[0110] For time dependent treatment, cells were transferred in
6-well plates with 3.times.10.sup.5 cells per well in 2 ml media.
Samples were prepared as detailed above or the single time point
experiments. Treatment was conducted at given time points prior to
harvest.
[0111] ML4924 and MG132 Treatment.
[0112] Cells were transferred in 6-well plates with
5.times.10.sup.5 cells per well in 2 ml media in order to achieve
80% confluence the day after. At t=0, MLN4924 was added into the
desired wells at 3 .mu.M final concentration and 0.1% v/v of DMSO.
DMSO (0.1% v/v final conc.) was added to the remaining wells in
order to match identical conc. of vehicle in all wells. At t=3 h,
MG 132 was added into the desired wells at 50 .mu.M final conc. and
0.1% v/v of DMSO. DMSO (0.1% v/v final conc.) was added to the
remaining wells in order to achieve the same conc. of vehicle in
all the wells. At t=3.5 h, the desired wells were treated with 1
.mu.M of CM11 in 0.1% v/v DMSO final concentration. DMSO (0.1% v/v
final conc.) was added to the remaining wells in order to obtain
the same conc. of vehicle in all the wells. The total final
concentration of DMSO was therefore 0.3% v/v. Plates were incubated
for 4 h at 37.degree. C. and 5% CO.sub.2 before harvesting.
[0113] Competition Experiments with VH032.
[0114] Cells were transferred in 6-well plates with
5.times.10.sup.5 cells per well in 2 ml media in order to achieve
80% confluence the day after. On the day of experiment, cells were
treated with VH032 at the final conc. of 150 .mu.M for 30 min prior
to treatment with CM11 at 1 .mu.M final concentration for 4 h.
Plates were incubated for the desired time at 37.degree. C. and 5%
CO.sub.2 before harvesting.
[0115] Co-Treatment with IOX4 and CM11 to Investigate Upstream
Effect Experiment.
[0116] For this experiments, cells were transferred in 6-well
plates with 5.times.10.sup.5 cells per well in 2 ml media in order
to achieve 80% confluence the day after. On the day of experiment,
cells were treated with IOX4 at the final concentration of 50 .mu.M
for 30 min prior to treatment with CM11 at 1 .mu.M final
concentration for 4 h. Plates were incubated for the desired time
at 37.degree. C. and 5% CO.sub.2 before harvesting.
[0117] Immunoblotting.
[0118] Cells were lysed in lysis buffer (20 mM Tris pH 8, 150 mM
NaCl, 1% Triton.times.100) and a protease inhibitor cocktail
(Roche) per 10 ml buffer. For protein extracts, the dishes were
placed on ice. The media was aspirated and the tissue layer washed
twice with ice-cold phosphate buffer saline (PBS). Lysis buffer
(120 pl) was added and the cells detached from the surface with a
cell scraper. After removal of the insoluble fraction by
centrifugation, the protein concentration of the supernatant was
determined by Pierce.TM. Coomassie (Bradford) Protein Assay Kit.
Protein extracts were fractionated by SDS-PAGE on 4-12%
Tris-Acetate NuPage.RTM. Novex.RTM. (Life Technologies)
polyacrylamide gels and transferred to a nitrocellulose membrane
using wet transfer. The membrane was then blocked with 5% w/v
Bovine serum albumin (BSA) in Tris-buffered saline (TBS) with 0.1%
w/v Tween-20. For detecting proteins the following primary
antibodies in the given concentrations were used: anti-.beta.-Actin
(Cell Signaling Technology, 4970S, 13E5) 1:2000, anti-VHL (Cell
Signaling Technology, #68547) 1:1000, anti-Hif-1.alpha. (BD
Biosciences, 610959, clone 54) 1:1000, anti-hydroxy-HIF-1.alpha.
(Hyp564) (Cell Signaling Technology; #3434) 1:1000, anti-PHD2
(Bethyl Laboratories; A300-322A) 1:1000, anti-PHD3 (Bethyl
Laboratories; A300-327A) 1:1000, anti-CRBN (Proteintech;
11435-1-AP) 1:1000.
[0119] Following incubation with a horseradish
peroxidase-conjugated secondary antibody (Cell Signaling
Technology), the signal was developed using enhanced
chemiluminescence (ECL) Western Blotting Detection Kit (Amersham)
on Amersham Hyperfilm ECL film (Amersham).
[0120] Band quantification was performed using ImageJ software and
reported as relative amount as ratio of the each protein band
relative to the lane's loading control. The values obtained were
then normalized to 0.1% DMSO vehicle control.
[0121] Luciferase Assay.
[0122] It was performed essentially as described by Frost et
al..sup.34 Briefly, cells (HeLa and U2OS) stably expressing an
HRE-luciferase reporter were treated for the indicated times with
compounds. Cells were harvested in passive lysis buffer (Promega)
and subjected to three freeze-thaw cycles. The soluble lysate
fraction was used for assays, performed according to the
manufacturer's instructions (Promega) using a Berthold Lumat LB
9507 Luminometer. Results were normalized for protein
concentration, and reported as mean.+-.s.e.m. from three biological
replicates.
[0123] Quantitative Real-Time PCR.
[0124] It was performed essentially as described by Frost et
al..sup.34 Briefly, RNA was extracted from HeLa cell lysates using
the RNeasy Mini Kit (Qiagen) and reverse transcribed using the
iScript cDNA Synthesis kit (Bio-Rad). Real-time PCR was performed
using PerfeCTa SYBR Green FastMix (Quanta Biosciences) in C1000
Touch Thermal Cycler (Bio-Rad). mRNA levels were calculated based
on averaged Ct values from two technical replicates, normalized to
mRNA levels of .beta.-actin, and reported as mean.+-.s.e.m. from
three biological replicates.
[0125] Biophysical Assays
[0126] Isothermal Titration Calorimetry (ITC).
[0127] Titrations were performed on an ITC200 micro-calorimeter (GE
Healthcare). PROTACs (CM11, CMP98 or CMP99) were diluted from a 100
mM DMSO stock solution to 150 .mu.M in a buffer containing 20 mM
Bis-tris propane, 150 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine
(TCEP), pH 7.4. The final DMSO concentration was 0.15% v/v. VBC
protein experiments were carried out in a buffer containing 20 mM
Bis-tris propane, 150 mM NaCl, 1 mM TCEP, 0.15% v/v DMSO, pH 7.4.
The titrations consisted of 19 injections of 2 .mu.L compounds
solution (150 .mu.M, in the syringe) at a rate of 2 s/.mu.L at 120
s time intervals into the VCB protein solution (20 .mu.M, in the
cell). An initial injection of compound solution (0.4 .mu.L) was
made and discarded during data analysis. All experiments were
performed at 25.degree. C., whilst stirring the syringe at 600 rpm.
The data were fitted to a single binding site model to obtain the
stoichiometry n, the dissociation constant K.sub.d and the enthalpy
of binding .DELTA.H using the Microcal LLC ITC200 Origin software
provided by the manufacturer.
[0128] Size Exclusion Chromatography (SEC).
[0129] SEC experiments were carried out in a AKTA pure system (GE
Healthcare) at room temperature. The oligomeric state of the VCB
complex in solution was analyzed by gel filtration in a buffer
containing 20 mM Bis-Tris (pH 7), 150 mM NaCl and 1 mM
1,4-dithiothreitol (DTT) using a Superdex 200 Increase 10/300 GL
column (GE Healthcare) calibrated with globular proteins of known
molecular weight (GE Healthcare, 28-4038-41/42). VBC protein (50
.mu.M) was incubated with CM11 (30 .mu.M), CMP98 (30 .mu.M), CMP99
(30 .mu..English Pound.M), VH032 (30 .mu.M) or DMSO (0.5%) for 20
min at room temperature prior to injection. Sample volume for each
injection was 200 .mu.L, and the flow rate was 0.5 mL/min. Peak
elution was monitored using ultraviolet absorbance at 280 nm.
[0130] Biotinylation of VCB.
[0131] The VCB complex was mixed with EZ-link NHS-PEG.sub.4-biotin
(Thermo Scientific) in a 1:1 molar ratio and incubated at room
temperature for 1 h. The reaction was quenched using 1 M Tris-HCl,
pH 7.5, and unreacted NHS-biotin was removed with a PD-10 MiniTrap
desalting column (GE Healthcare) equilibrated with 20 mM HEPES, pH
7.5, 150 mM NaCl and 1 mM DTT.
[0132] AlphaLISA Assay.
[0133] All assays were performed at room temperature in 384-well
plates with a final assay volume of 25 .mu.L per well; plates were
sealed with transparent film between addition of reagents. All
reagents were prepared as 5.times. stocks diluted in 50 mM HEPES,
pH 7.5, 100 mM NaCl, 0.1% (w/v) bovine serum albumin and 0.02%
(w/v) 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS). Biotinylated VCB (20 nM final) and His.sub.6-VCB (20 nM
final) were incubated with a range of Homo-PROTAC concentrations
(0.5 to 200 nM; three-in-five serial dilution) for 1 h. Anti-His
acceptor beads (PerkinElmer, 10 .mu.g/mL final) were added and
plates were incubated for another hour. Streptavidin-coated donor
beads (PerkinElmer, 10 .mu.g/mL final) were added and plates were
incubated for a final 1 h. Plates were read on a PHERAstar FS (BMG
Labtech) using an optic module with an excitation wavelength of 680
nm and emission wavelength of 615 nm. Intensity values were plotted
against PROTAC concentration on a logo scale.
[0134] Rational Design
[0135] Design of VHL Homo-PROTACs began with careful consideration
of the position of derivatization on two potent VHL ligands
recently characterized by our group, VH032 and VH298 (FIG.
1b)..sup.2,3 To retain the strong binding affinity that
characterizes the ligand, co-crystal structures were analyzed to
identify solvent exposed regions from where the ligands could be
derivatized without perturbing their binding modes (FIG. 1a). This
analysis and consideration of previous VHL-targeting PROTACs
pointed to the methyl group of the left-hand side (LHS) terminal
acetyl group of VH032 as a suitable point of connection for a
linker..sup.12,13 A second solvent-exposed position available for
derivatization was the phenyl group on the right-hand side (RHS),
as previously employed with PROTACs targeting the Halotag..sup.35
To investigate the impact of derivatization, we designed three
classes of Homo-PROTACS: a) symmetric via the LHS acetyl group of
each ligand (FIG. 2a); b) symmetric via the RHS phenyl group (FIG.
2b); and c) asymmetric via the acetyl group in one warhead and the
phenyl in the other (FIG. 2c). In the cases b and c, at the
underivatized terminal LHS we decided to retain either an acetyl
(as in VH032) or a cyano-cyclopropyl moiety (as in VH298), a
modification that led to increased binding affinities, cell
permeability and cellular activities in the context of the VHL
inhibitor alone..sup.3 To evaluate the potential impact of linker
length, linkers comprised of polyethylene glycol chains with either
three, four or five ethylene glycol units were chosen to connect
the two VHL ligands.
[0136] It is known that the trans epimer of Hyp is an absolute
requirement for VHL binding, and that the corresponding cis epimer
abrogates binding to VHL, both within the context of a native HIF
substrate peptide,.sup.36 and VHL ligands..sup.3,13 We therefore
designed two different PROTACs based on the structure of the first
series (FIG. 2a), with the aim to use them as controls: a cis-cis
epimer, expected to be completely inactive, and a cis-trans epimer
compound, expected to retain binding to a single VHL molecule in a
1:1 fashion, thus potentially acting as inhibitor but not as
degrader.
[0137] Synthesis.
[0138] For the synthesis of the first class of Homo-PROTACs (FIG.
2a), symmetric PEG linkers 4, 5 and 6 bearing free carbon/late
groups at either ends were obtained by reaction of tert-butyl
bromoacetate with tri-, tetra- and penta-ethylene glycol in the
presence of NaH in dioxane and followed, after purification, by
treatment with 50% TFA in DCM (FIG. 3). The final compounds CM9,
CM10 and CM11 were obtained by amide coupling of the VHL ligand 7
(prepared as previously described).sup.38 with linkers 4, 5 and 6,
in a 2:1 ratio, respectively, in the presence of HATU as the
coupling agent and DIPEA as the base (FIG. 3). For the synthesis of
the symmetric cis-cis compound CMP98, compound 8 (ref. 38) was
coupled with linker 6 to afford the desired product (FIG. 3).
[0139] For the preparation of the asymmetric cis-trans compound
CMP99, a synthetic route toward the synthesis of the monoprotected
di-carbon/late linker was established. Pentaethylene glycol was the
linker of choice because of ease of purification compared to longer
PEGs, and at the same time yielding a control compound of average
linker length (PEG-4 in this case). Pentaethylene glycol was
converted into monobenzyl ether 9 in 71% yield, which was reacted
with tert-butyl bromoacetic acid under biphasic conditions (DCM/37%
aq. NaOH and stoichiometric tetrabutyl ammonium bromide). After
deprotection of the benzyl group by catalytic hydrogenation,
formation of the carboxylic acid moiety was achieved by oxidation
with TEMPO and bis-acetoxy iodobenzene (BAIB), delivering compound
11 in 65% yield (FIG. 4). Compound 7 was then coupled with linker
11 using the condition described above, affording compound 12.
Deprotection of the tert-butyl group using TFA and subsequently
coupling with 8 afforded CMP99 in 66% yield (FIG. 4).
[0140] For the synthesis of the second class of symmetric
Homo-PROTACs (FIG. 2b), it was decided to utilize compounds 17 and
18 as VHL warheads. Common precursor 16 was synthesized following a
previously reported procedure,.sup.35 with minor modification that
led to yield and purity improvements (FIG. 5). Indeed, we observed
that the use of HATU in combination with HOAT for the coupling
steps of both Boc-L-Hyp and Boc-tert-leucine led to the formation
of only the desired products, avoiding the formation of a
bis-acylate secondary product,.sup.54 instead prominent when HATU
was used alone. Compound 17 or 18 were obtained by treatment of
compound 16 with 1-cyanocyclopropanecarboxylic acid in presence of
HATU, HOAT and DIPEA or acetylimidazole and TEA (FIG. 5). Synthesis
of 17 was also performed using acetic anhydride, but during this
reaction it was observed the formation of a secondary product
di-acetylated, not only at the desired position but also at the
hydroxyl group of the phenyl ring, which could however be
separated.
[0141] The PEG linkers for this class of compound were designed to
contain a methanesulfonate group at either end, which could be
coupled in a single step with the phenol of the VHL ligand. Linker
19 was prepared by mesylation of pentaethylene glycol and reacted
with either compounds 17 or 18 in a 1:2 ratio in the presence of
K.sub.2CO.sub.3 to afford CMP106 and CMP108, respectively, in good
yield (FIG. 6).
[0142] For the synthesis of asymmetric Homo-PROTACs, PEG 10 was
converted in to the mesylated derivative 20 and reacted with 17 or
18 to obtain 21 and 22, respectively in good yield (FIG. 7). Final
compounds CMP112 and CMP113 were obtained in good yield upon
deprotection of the tert-butyl group and amide coupling with
compound 7 (FIG. 7).
[0143] Biological Evaluation.
[0144] We next tested all our Homo-PROTACs in HeLa cells, and
monitored protein levels by Western blots after 10 h of compound
treatment at 1 .mu.M concentration (FIG. 8a). We observed striking
effectiveness of CM09, CM10 and CM11 in inducing VHL depletion in
cells (FIG. 8a), and a remarkably selective degradation for the
band corresponding to the long isoform of VHL, preferentially over
the short isoform. The VHL gene includes three exons and it encodes
two major isoforms of VHL: a 213 amino-acid long, 30 kDa form
(pVHL30) and a 160 amino-acid long, 19 kDa form (pVHL19). pVHL19
lacks a 53 amino-acid long amino-terminal domain or N-terminal tail
(pVHL-N), which is instead present in pVHL30. Although both
isoforms are expressed in human cells, pVHL19 is the more prominent
form in human tissues..sup.56 The most active compounds are
symmetrically linked from the terminal LHS acetyl group of VH032.
Linkage at different positions proved ineffective, suggesting a
critical role played by the linking pattern on the VHL ligands.
Control compounds CMP98 and CMP99 were unable to induce degradation
of VHL (FIG. 8a), demonstrating that Homo-PROTAC activity is
dependent on productive bivalent recruitment of VHL by the trans
epimer. The length of the linker also seemed to affect cellular
potency. Indeed, a decrease in effectiveness was observed at
shorter linker lengths, with CM10 and CM11 being the most active
compounds achieving total knockdown of pVHL30, followed by CM09
depleting 82% of the target protein. Interestingly, some
degradation of the short iso-form pVHL19 was also observed, albeit
low (around 10% depletion). Levels of Cullin2, the central subunit
of the CRL2-VHL complex,.sup.57 were also reduced upon treatment
with CM10 and CM11 by up to 22% (FIG. 8a).
[0145] Treatments with CM10 and CM11 also showed detectable albeit
low increase in protein levels of the hydroxylated form of
HIF-1.alpha. (Hdy-HIF-1.alpha., FIG. 8a). As the parent inhibitor
VH032 is completely ineffective at the same concentration of 1
.mu.M (see ref. .sup.3 and vide infra, FIG. 8), this effect cannot
be due to VHL inhibition and is therefore thought to be the result
of compound-induced protein degradation. Levels of HIF-1.alpha.
were, however, significantly lower than observed with the parent
inhibitors VH032 when used at concentrations >100 .mu.M (FIG.
8a, see also ref. .sup.3). VHL knockdown by siRNA experiments in
three different cell lines was consistent with CM11-induced
knockdown, and also insufficient to induce significant HIF
stabilization (FIG. 8b). The siRNA result also confirmed that the
bands observed to decrease in intensity with compound treatment
indeed correspond to VHL.
[0146] To assess whether selective pVHL19 knockdown by Homo-PROTACs
could induce HIF transcriptional activity, we first used a
luciferase reporter assay..sup.37 Hypoxia response element
(HRE)-luciferase reporter HeLa-HRE and U2OS-HRE cells were treated
with different concentrations of CM11 and at different times, and
no increase in HIF-dependent luciferase activity was detected
relative to DMSO control treatment (FIG. 14). These results were
confirmed in a qRT-PCR assay, where no upregulation of mRNA levels
of the known HIF-target genes CA9 was detected (FIG. 15). Together
the data suggests that the un-degraded pVHL19 is sufficient to
efficiently maintain low levels of HIF-1.alpha., and that complete
knockdown of all VHL isoforms is required to achieve effective HIF
stabilization in cells, as observed in vh.sup.-/- cells such as
VHL-deficient renal carcinoma cells.
[0147] We next turned our attention to further characterizing the
mode of action of the protein degradation induced by the active
Homo-PROTACs CM09-11. To interrogate their relative cellular
potency, dose-dependent treatments were performed at two different
time points, 4 and 24 h prior to harvesting. All compounds
confirmed preferential degradation of pVHL30 in a
concentration-dependent manner, relative to the corresponding DMSO
control (see FIG. 9 for CM11, and FIG. 16 for CM09 and CM10).
[0148] CM11 proved the most potent Homo-PROTAC, inducing complete
depletion of pVHL30 after 4 h already at 10 nM (DC.sub.99=10 nM,
FIG. 9). Selective pVHL30 knockdown was retained after 24 h, with
half-degrading concentration (DC.sub.50) between 10 and 100 nM. The
effective degrading concentrations of CM11 are >3 orders of
magnitude lower than the inhibitory concentrations of the
constitutive ligand VH032 alone, which is only active in cells at
.about.100 .mu.M, underscoring the profound difference in cellular
efficacy between the two mode of actions. Cellular levels of
Cullin2 decreased by up to 73% upon treatment with CM11 (FIG. 9).
As previously observed, selective pVHL30 knockdown by Homo-PROTACs
resulted in only minor increase in levels of HIF-1.alpha., relative
to hypoxia-inducing controls CoCl.sub.2, PHD inhibitor IOX2, and
VH032 (FIG. 9). However, when tested at high micromolar
concentrations, Homo-PROTACs acted preferentially as VHL inhibitors
over VHL degraders, consistent with the so-called "hook-effect"
whereby formation of binary 1:1 complexes competes with and
eventually supersedes the formation of the productive catalytic 2:1
complex..sup.59 Stabilization of Hdy-HIF-1.alpha. upon treatment
with all three compounds at 100 .mu.M was indeed comparable with
the effect obtained with VH032 alone (FIG. 9 for CM11, and FIG. 16
for CM09 and CM10). To confirm the cellular activities of
Homo-PROTACs in a different cell line, a similar experiment was
performed treating U2OS cells for 10 h with CM09, CM10 and CM11
using the same range of concentrations (1 nM-100 .mu.M). A
consistent profile of cellular activity was observed, confirming
that the effects observed are independent from cell type (FIG.
17).
[0149] We next interrogated the time-dependent activity of
Homo-PROTACs. Progressive removal of VHL protein over time was
observed, confirming selective depletion of pVHL30 over the short
isoform (FIG. 10 for CM11 and FIG. 18 for CM09 and CM10). In
particular, CM11 was confirmed to be the most effective compound,
decreasing pVHL30 level by more than 70% already after 2 h of
treatment, and essentially to completion after 8 h. The depletion
effect was retained up to 12 h; however, interestingly, pVHL30
levels up to 11% were detected after 24-36 h treatment, to then
decrease again after 48 h. Incomplete degradation of pVHL was
observed upon treatment with CM09, even in the longer time points
(FIG. 18). As before, minor stabilization of Hdy-HIF-1.alpha. over
time was observed for all three compounds, most pronouncedly up-on
treatment with CM11. The results obtained treating U2OS cells were
consistent with what observed in the previous experiment. However,
in this cell line all the three compounds were able to induce
complete degradation of pVHL30 over time (FIG. 17). We hypothesize
that this could be due to the lower expression level of VHL in
U2OS, leading to faster cellular depletion compared to cell lines
where VHL level is higher. CM09 and CM10 achieved complete
degradation of the target protein after 2 h of treatment. CM11
confirmed to be the most potent compound also in this cell line,
achieving complete degradation of pVHL30 already after 1 h.
Interestingly CM09 lost its cellular efficacy after 36 h. In
contrast, both CM10 and CM11 retained their efficacy even at these
longer time points (FIG. 17).
TABLE-US-00001 TABLE 1 Summary of thermodynamic binding parameters
of Homo-PROTACs and comparison with VHL inhibitor VH032 (from 19)
measured by ITC, against both short and long VHL isoforms.
-T.DELTA.S Protein Compound n Kd (nM) .alpha. .DELTA.G (kcal/mol)
.DELTA.H (kcal/mol) (kcal/mol) pVHL19 VH032 (ref. .sup.2) 1.030
.+-. 0.001 188 .+-. 6 -- -9.17 .+-. 0.02 -5.53 .+-. 0.01 -3.65 .+-.
0.02 CM11 0.6 .+-. 0.01 11 .+-. 2 18 -10.9 .+-. 0.1 -12.3 .+-. 0.7
1.4 .+-. 0.8 CMP99 0.964 .+-. 0.005 146 .+-. 2 -- -9.33 .+-. 0.06
-6.23 .+-. 0.05 -3.1 .+-. 0.7 CM09 0.98 .+-. 0.09 41 .+-. 15 4
-10.3 .+-. 0.2 -6.9 .+-. 0.3 -3.5 .+-. 0.5 CM10 0.73 .+-. 0.01 32
.+-. 5 6 -10.2 .+-. 0.1 -9.4 .+-. 0.1 -0.8 .+-. 0.2 CMP106 0.535
.+-. 0.004 111 .+-. 8 1.7 -9.5 -12.6 .+-. 0.1 3.1 CMP112 0.498 .+-.
0.006 235 .+-. 22 0.8 -9.1 -14.8 .+-. 0.2 5.8 CMP113 0.934 .+-.
0.005 117 .+-. 25 1.7 -9.5 -6.4 .+-. 0.2 -3.1 pVHL30 CM11 0.866
.+-. 0.003 25 .+-. 3 4 -10.4 .+-. 0.1 -11.3 .+-. 0.1 -0.9 .+-. 0.1
CMP99 1.050 .+-. 0.004 106 .+-. 10 -- -9.51 .+-. 0.05 -5.19 .+-.
0.03 -4.3 .+-. 0.1
[0150] To gain mechanistic insights in the cellular activity of
Homo-PROTACs, the dependency on CRL2-VHL and proteasome activities
was examined. The reliance of the Homo-PROTAC-induced protein
degradation on CRL2-VHL was assessed by inhibiting neddylation of
Cullin2 using the NAE1 inhibitor MLN4924, which blocks the activity
of CRLs, including CRL2-VHL. Proteasome-dependency was interrogated
by treating cells with the proteasome inhibitor MG132. To limit the
known cytotoxicity of MLN4924 and MG132, HeLa cells were
pre-treated with MLN4924 for 3 h followed by MG132 for 30 min
before adding CM11 to the media, and cells were incubated for
further 4 h before harvesting. Single treatments with DMSO,
MLN4924, MG132 and CM11 and combinations thereof were performed to
disentangle the individual and combined effects of compound
treatments. Degradation of pVHL30 induced by CM11 was completely
abrogated when cells were pre-treated with MG132, establishing the
expected proteasome-dependence of the chemical intervention (FIG.
11). CM11-induced degradation was also prevented by pre-treatment
with MLN4924, confirming the dependency on the activity of CRL2VHL
(FIG. 11). The same effect was observed when cells where co-treated
with MLN4924 and MG132 prior to CM11 (FIG. 11). Immunoblots of
Cullin2 levels confirmed the effective blockade of Cul2 neddylation
by MLN4924 (FIG. 11). To assess if CM11 de-grading activity was
dependent on VHL binding, a competition experiment was performed
using the VHL inhibitor VH032.20 HeLa cells were pre-treated with
VH032 at 150 .mu.M for 30 min before adding CM11 into the media.
The plates were incubated for further 4 h before harvesting. As
expected, VH032 blocked pVHL degradation (FIG. 11) consistent with
the hypothesis that VHL induces degradation of itself. In contrast,
pre-treatment with IOX4, a PHD2 inhibitor, did not impact the
cellular activity of CM11 (FIG. 11).
[0151] Biophysical Evaluation
[0152] Key to the catalytic mode of action of PROTACs is the
formation of a ternary complex..sup.13,15 In the case of our
Homo-PROTAC compounds, VHL acts as both the E3 ligase and the
substrate. Therefore, we next sought to monitor and biophysically
characterize the ternary complex VHL:Homo-PROTAC:VHL that is
thought to underlie cellular activity. To assess the formation of
this ternary complex species in solution, isothermal titration
calorimetry (ITC), size exclusion chromatography (SEC) and
AlphaLISA proximity assays were performed (FIG. 12). In ITC
titration of CM11 against the VCB complex (VHL with Elongin B and
Elongin C) the stoichiometry of binding (n value) was found to be
0.6, instead of 1 with VH032 (FIG. 12a, Table 1). This result is
consistent with CM11 binding to VHL in a 1:2 molar ratio, in
contrast to VH032 that binds to VHL in a 1:1 ratio..sup.19 Notably,
the K.sub.d value measured for CM11 was 11 nM (Table 1). Closer
examination of the titration curve revealed that only one point
features during the inflection of the curve. Indeed, because the
protein concentration used in the experiment was 20 .mu.M, the c
value (defined as [P].sub.tot/K.sub.d) calculated for this
experiment is 2500, which is well above the upper limit of c
(around 500-1000) that is a prerequisite for precise measurement of
binding affinity. Consequently, this analysis suggests that we may
be underestimating the binding affinity of CM11, i.e. we can
conclude that K.sub.d is .ltoreq.118 nM. This corresponds to an
avidity (also known as cooperativity .alpha.) of >18-fold when
compared to VH032. Such large avidity of homobivalent molecules has
been observed previously with other systems, for example the BET
inhibitor MT1. The binding interaction between CM11 and VHL was
driven by a large apparent binding enthalpy (.DELTA.H=-12.3 kcal
mol.sup.-1), whereas the entropic term was slightly unfavourable
(-T.DELTA.S=1.4 kcal mol.sup.-1). This observation underlines how
the thermodynamic signature of CM11 is also very different when
compared with that of VH032, in which case the binding .DELTA.H was
around half that observed with CM11, and both the enthalpic and
entropic term contributed favourably to the .DELTA.G of binding
(Table 1). By contrast, the thermodynamic values obtained for CMP99
binding were entirely consistent with the ones of VH032 (Table 1).
Specifically, CMP99 bound to VHL in a 1:1 ratio, as expected due to
the presence of the cis-Hyp in one of the two moieties, and it
exhibited comparable .DELTA.H and K.sub.d values to VH032. As
expected, binding was not detected with CMP98, the inactive cis-cis
epimer. Superposition of integrated heat curves of CM11, CMP98 and
CMP99 is shown in FIG. 12b and visually highlights the different
behaviours of the three compounds. CM10 showed similar
thermodynamic binding parameters relative to CM11, with n value
equal to 0.7 and a low K.sub.d of 32 nM. A stoichiometry close to 1
was instead found for CM09, suggesting that at the end of the
titration this system was primarily populated by 1:1 complexes
(FIGS. 19 and 20), consistent with its lower avidity (Table 1). ITC
experiments were also conducted with compounds CMP106, CMP108,
CMP112 and CMP113, and the results are discussed below.
[0153] SEC experiments showed that VCB migrates more quickly in the
presence of the active compound CM11 (2:1 protein:ligand ratio),
relative to the vehicle control (FIG. 12b). The shifted peak eluted
at a volume corresponding to a species of .about.90 kDa molecular
weight, based on a calibration run with globular proteins of known
molecular weight (see Methods below), suggesting the peak
corresponds to the ternary complex (VCB).sub.2:CM11. In contrast,
there is no shift in VCB following incubation with inactive CMP98,
CMP99 or ligand VH032. Only in the sample containing CMP99 a small
peak eluted at 13.5 ml (FIG. 12b). It is possible that such peak
could be due to the formation of a lowly populated ternary complex.
It is interesting that Schofield and colleagues observed weak
binding of a cis-hydroxyprolyl containing HIF-1.alpha. peptide to
VHL..sup.36 This weak binding, potentially enhanced by high avidity
in the ternary complex, could be responsible for the small decrease
of VHL levels observed during biological tests in cells (FIG. 8a).
CM10 and CM09 showed formation of a ternary complex eluting at
identical retention volume when compared to CM11 (FIG. 21). No
evidence of aggregation was seen with any of the compounds
evaluated, as all observed peaks eluted well after the void
volume.
[0154] Lastly, we employed an AlphaLISA proximity assay to compare
ternary complex formation by CM09, CM10 and CM11. The assay showed
the highest intensity signal for CM11, whereas negligible levels of
complex formation were detected for CM09 and CM10 (FIG. 12c). Since
SEC detected ternary species with all three compounds, the minimal
intensity detected in the AlphaLISA likely reflects the inability
of CM09 and CM10 to yield a significant ternary population at the
low concentrations required for the assay. These results indicate
that CM11 is the most effective Homo-PROTAC at driving ternary
complex formation, consistent with CM11 exhibiting the highest
avidity and full 2:1 stoichiometry in ITC. Together, the
biophysical data supports CM11 as the most cooperative Homo-PROTAC
in vitro, and provide a molecular rationale explaining its potent
VHL-degrading activity inside cells.
[0155] Discussion
[0156] In some embodiments, Homo-PROTACs are described, a
small-molecule approach to effectively dimerize an E3 ubiquitin
ligase to induce its own self-destruction. Using potent ligands for
the E3 ligase VHL, a series of symmetric homo-bivalent molecules
that induce remarkably rapid, profound and selective degradation of
the long isoform of pVHL at nanomolar concentrations were
developed. Compound-induced degradation was exquisitely dependent
on the linkage pattern on the VHL ligand. The most active
Homo-PROTAC, CM11, induces complete depletion of pVHL30 after 4 h
already at 10 nM. Potent and selective degradation of pVHL30 was
long lasting, with half-degrading concentration (DC.sub.50) of
approximately 100 nM, a remarkable increase in cellular activity of
>1000-fold compared to the parent inhibitor VH032.
Mechanistically, it has been shown that CM11 activity is strictly
dependent on proteasome activity, Cul2 neddylation, and on VHL
binding, and specifically on the formation of an avid 2:1 complex
with VHL. The data therefore supports a model in which a highly
cooperative ternary complex VHL-CM11-VHL functions as the key
species responsible for the induced degradation of VHL itself (FIG.
13), which will warrant future structural studies. Interestingly,
CM11 also led to a decrease in cellular levels of Cullin2, which we
hypothesize to be the result of direct ubiquitination of Cullin2 as
part of the CRL2vHL complex. To our knowledge, this is first
demonstration that a PROTAC can induce the degradation of a protein
forming part of the same complex with the protein targeted
directly.
[0157] The preferential induced degradation of pVHL30 over the
short VHL isoform was unexpected and is an intriguing result of
this work. This observation adds to recent evidence from us and
others that chemical degraders designed from inhibitors recruiting
more than a single protein paralog or isoform can add a layer of
target degradation selectivity independently of target
engagement..sup.12,15,18 As the binary engagement of the VHL
warhead was found to be similar between the two VHL isoforms (Table
1), the observed selectivity could be due to large differences in
cooperativities, which would impact on the relative population of
ternary complexes..sup.15 However, CM11 actually exhibited greater
avidity in vitro for the short relative to the long isoform of VHL
(Table 1). We therefore view it as unlikely that the remarkable
selectivity of VHL degradation is due to large differences in
cooperativities of ternary complexes. We also consider unlikely
that preferential and more efficient lysine ubiquitination could
play a role, because the extra region present in the long isoform
(1-53) does not contain a single lysine residue. On the other hand,
this region is predicted as intrinsically disordered, and indeed it
has been shown that proteins containing disordered N-terminal
regions are more prone to proteasomal degradation. It is also known
that VHL is resistant to proteasomal degradation when in complex
with ElonginB and ElonginC, so the form observed to be
preferentially depleted may be free VHL i.e. unbound to Elongins,
or other proteasome-sensitive forms. Addressing these questions
will be of clear importance for future investigation.
[0158] Selective degradation of pVHL30 by CM11 led to minimal
stabilization of HIF-a in cells, and as a result did not trigger
HIF-dependent activity in cells. This highlights the potential
benefit of using CM11 to interrogate the biological function of
specific VHL isoforms, without the masking downstream effects of a
hypoxic response. Not much is known about the individual roles of
VHL isoforms. Studies have highlighted how the 53-residue extra
region of pVHL30 is not needed for tumor suppression, and how both
isoforms can have HIF-dependent tumor suppressor functions in vivo.
Other HIF-independent roles of pVHL have been proposed, including a
role for pVHL in collagen assembly. However, the individual roles
of the different isoforms in these biological functions remain
elusive. Moreover, many HIF-independent roles are thought to be
independent upon Hyp recognition, and thus cannot be probed
chemically using current Hyp-based VHL inhibitors. Selective and
acute knockdown of pVHL30 by CM11 provides therefore a novel
chemical tool to address these questions.
[0159] In summary, we present CM11, a chemical probe for rapid and
selective pVHL30 knockdown. CM11 provides an alternative
advantageous chemical tool to conventional knockdown RNAi
approaches and gene editing knockout technologies such as
CRISPR-Cas9. Relevant information to the use of CM11 will be made
available in the newly established "Chemical Probes Portal"
(http://www.chemicalprobes.org/)..sup.38 We anticipate CM11 will
find wide use amongst chemical and cell biologists alike interested
in investigating and dissecting the pleiotropic biological
functions of pVHL. More generally, we provide first
proof-of-concept that bivalent molecules can be designed to induce
an E3 ligase to destroy itself. This strategy could provide a
powerful new approach to drugging E3 ligases in ways that may not
be possible with inhibitors alone.
[0160] Synthesis of PROTACs Recruiting Together CRL4.sup.CRBN and
CRL2.sup.VHL.
[0161] For the synthesis of compounds CMP85 and CMP86 (structures
shown in FIG. 23), the linker 26 and its analogue with two PEG
units 43 were synthesized adopting the same route used for 26 (FIG.
24). These linkers were then coupled to compound 27, delivering
compounds 28 and 44, respectively. Subsequent deprotection of the
tert-butyl group afforded compound 29, with a length of four PEG
units, and 45, with two PEG units instead (FIG. 24).
[0162] Compound 48 (the desired thalidomide derivative, see Figure)
was synthesized as previously published by Lu et al..sup.17 In the
first step, 3-fluorophthalic acid was dehydrated with acetic
anhydride to obtain compound 46 in good yield. Reaction of compound
46 with L-glutamine and subsequent treatment with HCl 4 M solution
led to the formation of compound 47. Cyclization of 47 was
performed at reflux in the presence of 1,1'-carbonyldiimidazole
(CDI) and DMAP. The recommended time for this step was 5 h. After
2.5 h it was possible to observe the formation of a side product by
LC-MS. For this reason, even if the reaction was not completed, the
reaction was cooled to r.t. and the resulting solid collected by
filtration. During the purification step, performed by column
chromatography over silica, compound 48 was isolated in good yield.
The side product was isolated as well and analysed by NMR and
identified to be compound 53 (FIG. 26). Compound 53 is the product
of an aromatic nucleophilic substitution at position 4 of the
phthalic anhydride by the nitrogen lone pair of imidazole, which is
itself a byproduct of the reaction between 47 and CDI.
[0163] Compound 48 was converted into compound 50 in two steps
(FIG. 25), by coupling with N-Boc-ethylenediamine and subsequent
Boc deprotection in acidic conditions. Coupling of the latter with
29 or 45 afforded compounds 52 (CMP85) and 51 (CMP86) respectively
in good yield.
[0164] Biological Evaluation of the VHL-Targeting Compounds
[0165] The following section outlines the results of the biological
evaluation of PROTAC compounds targeting VHL in cells.
[0166] In order to assess the activity of compounds inside cells,
HeLa cells were treated with 1 .mu.M of Homo-PROTACs CM09, CM10 and
CM11 (FIG. 27) synthesis of which are below, and the above
described PROTACs recruiting CRL4.sup.CRBN to target VHL, i.e.
CMP85 and CMP86.
[0167] Dimethylsulfoxide (DMSO vehicle, 0.1% v/v), CoCl.sub.2
(chemical inducer of HIF-1.alpha.), IOX2 and IOX4 (selective
inhibitor of PHD2), VH032 (selective VHL inhibitor) were used as
controls. The samples, obtained after 10 h of treatment and cell
lysis, were resolved by SDS-PAGE followed by Western blot using the
corresponding specific antibodies to probe for the following
proteins (FIG. 28): [0168] VHL: CM09, CM10 and CM11 demonstrated
complete depletion of VHL levels, which featured as a preferential
or selective degradation of the long isoform pVHL30. However, some
degradation of the short isoform pVHL19 was also observed, albeit
only around 20%. None of the other compounds were able to induce
degradation of VHL. [0169] Cullin2: To assess if treatment with the
series of compounds could have any effect on other subunits of the
CLR2.sup.VHL, protein levels of Cullin2 were evaluated. CM10 and
CM11 showed to affect Cullin2 levels by inducing a reduction of
approximately 20%. [0170] CRBN: No detectable effect was observed
on CRBN levels upon treatment with CMP85 and CMP86. [0171]
HIF-1.alpha. and Hdy-HIF-1.alpha.. To evaluate if VHL degraders
could induce accumulation of HIF-1.alpha., and specifically of its
hydroxylated form (Hdy-HIF-1.alpha.), levels of these proteins were
evaluated. It was observed during siRNA experiments that VHL
knockdown does not lead to HIF-1.alpha. depletion. Indeed, even
very low levels of VHL are capable of highly efficient catalysis on
HIF-1.alpha., leading to subsequent effective HIF-1.alpha.
degradation. As expected, VHL depletion did not impact
significantly on HIF-1.alpha. level (compare the detected
HIF-1.alpha. band with vehicle control DMSO). Nevertheless, a
slight increase of HIF-1.alpha. level was induced by the active VHL
degraders CM09, CM10 and CM11 (see HIF-1.alpha. band with longer
exposure). This effect was even more pronounced on
Hdy-HIF-1.alpha., consistent with the stabilized HIF being in the
hydroxylated form as expected from VHL knockdown. [0172] PHD2 and
PHD3: to study potential hypoxic response of cells due to treatment
with the compounds, levels of PHD2 and PHD3 were considered. No
effect on the levels of these proteins was observed at this
concentration.
[0173] The same experiments were performed in other cells lines to
further assess the consistency of the cellular effects of our
compounds, as different cell lines can have different expression
levels of different proteins. For example, HEK293 are known to have
higher expression levels of total VHL, which we confirmed by
Western blot (FIG. 28). The same activity profile in decreasing
preferentially pVHL30 levels by CM09-11 was observed in HEK 293
(FIG. 28). No major effects were observed on levels of the other
proteins. Experiments conducted in U2OS cells showed the same
results, confirming that the effect observed upon treatment with
CM09, CM10 and CM11 is independent from cell type and it is
consistent in all tested cell lines (FIG. 28).
[0174] ITC experiments were also conducted with compounds CMP106,
CMP108, CMP112 and CMP113 (data shown in FIGS. 29-32). With the
exception of compound CMP108 for which data could not be fitted to
a binding curve, all of the other compounds exhibited very similar
binding affinity as the individual warhead ligand (cooperativity
around 1), suggesting they are much less cooperative than CM11,
which is consistent with their lack of activity in cells. This
conclusion is illustrated in FIG. 33, where the ITC titrations for
compounds CM09-11, CMP112-113, CMP106 as well as control compounds
CMP98 and CMP99 are all superposed together in a single Figure,
highlighting the remarkable potency and cooperativity of CM11.
[0175] Materials and Methods
[0176] All chemicals were purchased from commercial vendors and
used without further purification, unless indicated otherwise.
Reactions were magnetically stirred; commercially available
anhydrous solvents were used. All reactions requiring anhydrous
conditions were carried out under argon or nitrogen atmosphere
using oven-dried glassware. HPLC-grade solvents were used for all
reactions. Flash column chromatography was carried out using silica
gel (Merck 60 F254 nm). Normal phase TLC was carried out on
pre-coated silica plates (Kieselgel 60 F254, BDH) with
visualization via UV light (UV 254/365 nm) and/or basic potassium
permanganate solution or other suitable stains. Flash column
chromatography (FCC) was performed using a Teledyne Isco Combiflash
Rf or Rf200i, prepacked columns RediSep Rf Normal Phase Disposable
Columns were used. NMR spectra were recorded on a Bruker Ascend 400
or 500. Chemical shifts are reported in parts per million
referenced to residual solvent peaks (CDCl.sub.3=7.26 ppm). The
following abbreviations were used in reporting spectra, s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd
(doublet of doublets). Only major rotamer NMR spectra are reported.
High Resolution Mass Spectra (HRMS) were recorded on a Bruker
microTOF. Low resolution MS and analytical HPLC traces were
recorded on an Agilent Technologies 1200 series HPLC connected to
an Agilent Technologies 6130 quadrupole LC/MS, connected to an
Agilent diode array detector. The column used was a Waters XBridge
column (50 mm.times.2.1 mm, 3.5 .mu.m particle size). The flow rate
was 0.6 mL/min. Preparative HPLC was performed on a Gilson
Preparative HPLC System with a Waters XBridge C18 column (100
mm.times.19 mm; 5 .mu.m particle size).
[0177] General method A: PEG was solubilised in dioxane anhydrous
and NaH was added under stirring. The resulting mixture was stirred
at r.t. for 3 h. The mixture was cooled down to 0.degree. C. using
ice bath and tert-butylbromo acetate was added drop by drop. The
resulting mixture was stirred at r.t O/N. The precipitate was
filtered off and the organic phase evaporated to dryness. The
resulting oil was taken up with ethyl acetate, washed with water,
dried over MgSO.sub.4 and evaporated to dryness. The resulting oil
was purified by column chromatography using a gradient of ethyl
acetate from 50% to 100% v/v in heptane.
[0178] General method B: tert-butyl esters 1, 2, 3 or 12 were
dissolved in a solution of 50% v/v trifluroacetic acid in DCM. The
resulting solution was stirred for 1 h or until complete conversion
of starting material. The solvent was removed under high vacuum.
The resulting carboxylic acid was used as crude in the next step
without any further purification. To a solution of carboxylic acid
in 1 ml DMF were added HATU (1 eq.) and HOBT (1 eq.) and the
solution was stirred at room temperature for 5 min. Amine 6, 31 or
32 was added and the pH of the reaction mixture was adjusted to
>9 by addition of DIPEA (3 eq.). The mixture was stirred at room
temperature until no presence of the starting materials was
detected by LC-MS. Water was added and the mixture was extracted
with ethyl acetate (.times.3). The combined organic phases were
washed with brine (.times.2), dried over MgSO.sub.4 and evaporated
under reduced pressure to give the corresponding crude, which was
purified by HPLC using a gradient of 20% to 95% v/v acetonitrile in
0.1% aqueous solution of ammonia to yield the desired compound.
[0179] General method C: A mixture of mesilate, compound 6, 31, 32,
and K.sub.2CO.sub.3 (41.46 mg, 0.3 mmol, 6 eq.) in DMF was stirred
O/N at 70.degree. C. The reaction mixture was filtered off to
afford the crude product, which was purified by HPLC using a
gradient of 5% to 95% v/v acetonitrile in 0.1% aqueous solution of
formic acid to yield the desired compounds.
(2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine--
2-carboxamide hydrochloride (15)
##STR00005##
[0181] To a solution of
trans-N-(tert-Butoxycarbonyl)-4-hydroxy-L-proline (890 mg, 3.84
mmol, 1 eq.) in DMF was added HATU (1.46 g, 3.84 mmol, 1 eq.) and
HAOT (522 mg, 3.84 mmol, 1 eq.) and the solution was stirred at
room temperature for 5 min. 14 (846 mg, 3.84 mmol, 1 eq.) was added
and the pH of the reaction mixture was adjusted to >9 by
addition of DIPEA (3 eq.) and the mixture was stirred at room
temperature until no presence of the starting materials was
detected by LC-MS. Water was added and the mixture was extracted
with ethyl acetate (.times.3). The combined organic phases were
washed with brine (.times.2), dried over MgSO.sub.4 and evaporated
under reduced pressure to give the corresponding crude, which was
purified by flash column chromatography using a gradient of 0 to
80% v/v acetone in heptane to yield the titled compound. Yield:
1.298 g, 3 mmol (78%). Analytical data matched those previously
reported.sup.35
[0182] The N-Boc-protected compound was dissolved in DCM. An equal
volume of 4M HCl in dioxane was added and the reaction mixture
stirred at room temperature for 2 h. The solvent was removed under
a stream of nitrogen and dried under reduce pressure. The resulting
crude was used for the next step without any further purification
(quantitative yield). Analytical data matched those previously
reported.sup.35
(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-m-
ethylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride
(16)
##STR00006##
[0184] To a solution of
trans-N-(tert-Butoxycarbonyl)-4-hydroxy-L-proline (890 mg, 3.84
mmol, 1 eq.) in DMF was added HATU (1.46 g, 3.84 mmol, 1 eq.) and
HAOT (522 mg, 3.84 mmol, 1 eq.) and the solution was stirred at
room temperature for 5 min. 14 (846 mg, 3.84 mmol, 1 eq.) was added
and the pH of the reaction mixture was adjusted to >9 by
addition of DIPEA (3 eq.) and the mixture was stirred at room
temperature until no presence of the starting materials was
detected by LC-MS. Water was added and the mixture was extracted
with ethyl acetate (.times.3). The combined organic phases were
washed with brine (.times.2), dried over MgSO.sub.4 and evaporated
under reduced pressure to give the corresponding crude, which was
purified by flash column chromatography using a gradient of 0 to
80% v/v acetone in heptane to yield the titled compound. Yield:
1.915 g, 3.61 mmol (94%). Analytical data matched those previously
reported.sup.35.
[0185] The N-Boc-protected compound was dissolved in DCM. An equal
volume of 4 M HCl in dioxane was added and the reaction mixture
stirred at room temperature for 2 h. The solvent was removed under
a stream of nitrogen and dried under reduced pressure. The
resulting crude was used for the next step without any further
purification (quantitative yield). Analytical data matched those
previously reported.sup.35.
(2S,4R)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-
-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carbo-
xamide (18)
##STR00007##
[0187] 1-cyanocyclopropanecarboxylic acid (69 mg, 0.62 mmol, 1 eq.)
was solubilized in 3 ml of DMF. HATU (235 mg, 0.62 mmol, 1 eq.) and
HOAT (84.4 mg, 0.62 mmol, 1 eq.) were added and the resulting
mixture was stirred at r.t. for 5 min. The amine precursor of 16
(300 mg, 0.62 mmol, 1 eq.) was added and the pH was adjusted to
pH>9 using DIPEA (400 mg, 0.5 ml, 3.1 mmol, 5 eq.). The
resulting mixture was stirred at r.t. until complete conversion of
the starting material. Water was added, and the mixture was
extracted with ethyl acetate (.times.3). The combined organic
phases were washed with brine (.times.1), dried over MgSO.sub.4,
and evaporated to afford the corresponding crude compound that was
purified by flash column chromatography using a gradient of 10% to
70% acetone in heptane to yield the title compound as a white
solid. Yield: 200 mg, 0.37 mmol (60%). HRMS (ESI) m/z: [M+H].sup.+
calculated for: C.sub.27H.sub.33N.sub.5O.sub.5S: 539.22; observed:
540.3.
[0188] .sup.1H NMR (400 MHz, CDCl3) 9.29 (1H, s), 8.65 (1H, s),
8.02 (1H, t, J=6.4 Hz), 7.12 (1H, d, J=7.7 Hz), 6.99 (1H, d, J=8.0
Hz), 6.94 (1H, d, J=1.8 Hz), 6.86 (1H, dd, J=1.8, 7.7 Hz), 4.72
(1H, t, J=8.0 Hz), 4.54 (1H, s), 4.44-4.35 (2H, m), 4.19 (1H, dd,
J=5.5, 14.6 Hz), 3.87 (1H, d, J=11.0 Hz), 3.62 (1H, dd, J=3.7, 11.0
Hz), 3.50 (1H, s), 2.49 (3H, s), 2.43-2.37 (1H, m), 2.13-2.06 (1H,
m), 1.66-1.37 (4H, m), 0.89 (8H, s); .sup.13C NMR (101 MHz,
CDCl.sub.3) .delta. 172.8, 170.8, 165.8, 155.8, 150.5, 148.3,
133.3, 131.6, 131.2, 123.9, 120.9, 119.6, 118.2, 70.1, 58.6, 58.3,
56.7, 55.7, 40.0, 35.7, 26.2, 18.6, 17.9, 17.8, 17.2, 16.1,
13.8.
(2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4--
(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17)
##STR00008##
[0190] The amine precursor 16 (100,7 mg, 0.240 mmol, 1 eq.) was
dissolved in 1 ml of DMF, acetylimidazole (31.7 mg, 0.288 mmol, 1.2
eq) and DIPEA (0.090 ml, 0.48 mmol, 2 eq.) were added to the
solution. After stirring the mixture for 48 h at room temperature,
the solvent was evaporated under reduced pressure to give the
corresponding crude, which was purified by HPLC using a gradient of
5% to 95% v/v acetonitrile in 0.1% aqueous solution of formic acid
to yield the titled compound. Yield: 91 mg, 0.187 mmol (78%).
.sup.1H NMR (400 MHz, CDCl3) 9.25 (1H, s), 8.70 (1H, s), 7.97 (1H,
t, J=6.5 Hz), 7.15 (1H, d, J=7.5 Hz), 6.83-6.80 (2H, m), 6.72 (1H,
d, J=8.8 Hz), 4.92-4.88 (1H, m), 4.57 (1H, s), 4.52-4.42 (2H, m),
4.26-4.14 (2H, m), 3.59 (1H, dd, J=2.9, 11.1 Hz), 2.53-2.45 (4H,
m), 2.24-2.17 (1H, m), 1.85 (3H, s), 0.83 (9H, s); .sup.13C NMR
(101 MHz, CDCl.sub.3) .delta. 171.8, 171.2, 155.9, 150.7, 148.1,
132.8, 131.7, 131.0, 124.2, 120.6, 117.1, 70.3, 58.1, 57.7, 57.1,
39.8, 35.5, 34.8, 26.3, 22.6, 16.0. HRMS (ESI) m/z: [M+H].sup.+
calculated for: C.sub.24H.sub.32N.sub.4O.sub.5S: 488.21; observed:
484.3.
di-tert-butyl 3,6,9,12-tetraoxatetradecanedioate (1)
##STR00009##
[0192] Following general method A, from triethylene glycol (1.125
g, 1 ml, 7.49 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral
oil (595.75 mg, 14.9 mmol, 2 eq.) and tert-Butyl bromoacetate
(2.905 g, 2.19 ml, 14.9 mmol, 2 eq.), compound 1 was obtained as an
oil after high vacuum. Yield: 538 mg, 1.42 mmol (19%).
[0193] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 3.81 (4H, s),
3.51-3.46 (12H, m), 1.26 (18H, s). .sup.13C NMR (126 MHz,
CDCl.sub.3) .delta. 169.1, 80.9, 70.1, 70.0, 68.5, 27.5. Analytical
data matched those previously reported. .sup.39
di-tert-butyl 3,6,9,12,15-pentaoxaheptadecanedioate (2)
##STR00010##
[0195] Following general method A, from tetrathylene glycol (1.125
g, 1 ml, 5.49 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral
oil (463 mg, 11.5 mmol, 2 eq.) and tert-Butyl bromoacetate (2.25 g,
1.7 ml, 11.5 mmol, 2 eq.), compound 2 was obtained as an oil after
high vacuum. Yield: 500 mg, 1.18 mmol (10%).
[0196] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 3.86 (4H, s),
3.55-3.49 (16H, m), 1.31 (9H, s). Analytical data matched those
previously reported..sup.39
di-tert-butyl 3,6,9,12,15,18-hexaoxaicosanedioate (3)
##STR00011##
[0198] Following general method A, from pentaethylene glycol (1.126
g, 1 ml, 4.72 mmol, 1 eq.) in 10 ml of dioxane, NaH 60% in mineral
oil (377 mg, 9.45 mmol, 2 eq.) and tert-Butyl bromoacetate (1.872
g, 1.7 ml, 11.5 mmol, 2 eq.), compound 3 was obtained as an oil
after high vacuum. Yield: 300 mg, 0.641 mmol (14%).
[0199] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.94 (4H, s),
3.66-3.56 (20H, m), 1.40 (18H, s). Analytical data matched those
previously reported.sup.39.
1-phenyl-2,5,8,11,14-pentaoxahexadecan-16-ol (9)
##STR00012##
[0201] Pentaethylene glycol (9.53 g, 50 mmol, 5 eq.) was added
dropwise to a suspension of NaH 60% in mineral oil (800 mg, 20
mmol, 2.5 eq.) in 20 ml of DMF at 0.degree. C. The resulting
mixture was stirred at r.t for 1 h. The reaction mixture was cooled
to 0.degree. C., benzyl chloride (1 ml, 1.1 g, 8.72 mmol, 1 eq.)
was added. The resulting mixture was stirred O/N at r.t. The
reaction was quenched with a saturated solution of NH.sub.4Cl and
the aqueous phase was extracted with ethyl acetate (.times.3). The
combined organic phases were dried over MgSO.sub.4 and evaporated
to dryness. The resulting oil was purified by column chromatography
(from 0 to 60% of ethyl acetate in heptane) to afford the title
compound as an oil. Yield: 2.055 g, 6.25 mmol (71%).
[0202] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.28-7.19 (5H, m),
4.50 (2H, s), 3.66-3.52 (20H, m), 2.50 (1H, 5). .sup.13C NMR (101
MHz, CDCl.sub.3) 138.2, 128.3, 127.8, 127.6, 73.2, 72.7, 70.61,
70.58, 70.53, 70.51, 70.2, 69.4, 61.7
tert-butyl 1-phenyl-2,5,8,11,14,17-hexaoxanonadecan-19-oate
(10)
##STR00013##
[0204] To a stirred solution of 9 (2.055 g, 6.25 mmol, 1 eq.) in
12.8 ml of DCM was added 37% solution of NaOH (12.8 ml), followed
by tert-butylbromo acetate (4.882 g, 25 mmol, 4 eq.) and TBABr
(2118 mg, 6.37 mmol, 1.02 eq.). The resulting solution was stirred
O/N at r.t. The reaction mixture was extracted with ethyl acetate
(.times.3). The organic phases were combined and washed with brine
(.times.1), dried over MgSO.sub.4 and concentrate in vacuo. The
resulting brown oil was purified by column chromatography (from 0
to 30% of ethyl acetate in petroleum) to afford the titled compound
as colourless oil. Yield: 2.216 g, 5 mmol (80%).
[0205] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.28-7.20 (5H, m),
4.50 (2H, s), 3.95 (2H, s), 3.65-3.55 (20H, m), 1.40 (9H, s).
.sup.13C NMR (126 MHz, CDCl.sub.3) .delta.169.7, 128.4, 127.7,
127.6, 81.5, 73.2, 70.7, 70.7, 70.6, 70.6, 69.4, 69.1, 28.1. HRMS
(ESI) m/z: [M+H].sup.+ calculated for: C.sub.23H.sub.38O.sub.8:
442.26; observed: 387.2.
19,19-dimethyl-17-oxo-3,6,9,12,15,18-hexaoxaicosanoic acid (11)
##STR00014##
[0207] 10 (2.216 g, 5 mmol, 1 eq.) was dissolved in 75 ml of
ethanol, Pd/C (10 wt %) was added and the resulting mixture was
place under hydrogen and stirred at r.t. until complete conversion
of the starting material. The reaction mixture was filtered through
celite, the celite pad was washed few times using ethanol. The
filtrate was concentrated in vacuum to give an oil that was used
for the next step without further purification. Yield: 1.764 g, 5
mmol (quantitative).
[0208] BAIB (3.546 g, 11.01 mmol, 2.2 eq.) and TEMPO (171.87 mg,
1.10 mmol, 0.22 eq.) were added to a solution of ACN/H.sub.2O 1:1
containing previous obtained oil (1.764 g, 5 mmol, 1 eq.). The
resulting mixture was stirred at r.t until complete conversion of
the starting material. The crude was purified using ISOLUTE.RTM.
PE-AX anion exchange column. The column was equilibrate with
methanol, the reaction mixture poured in the column and let it
adsorbed in the pad. The column was washed with methanol (.times.3)
to elute all the unbound material. Then, the titled product was
eluted using a 50% solution of formic acid in methanol. The organic
phase was evaporated to dryness to afford the title compound as
oil. Yield: 1.200 g, 3.27 mmol (65%).
[0209] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta., ppm 4.12 (2H, s),
3.98 (2H, s), 3.72-3.60 (16H, m), 1.43 (9H, s). .sup.13C NMR (101
MHz, CDCl.sub.3) .delta., ppm 172.6, 169.7, 81.6, 71.0, 70.59,
70.56, 70.54, 70.46, 70.38, 70.35, 70.30, 68.9, 68.8, 28.1.
3, 6,9,12-tetraoxatetradecane-1,14-diyl dimethanesulfonate (19)
##STR00015##
[0211] Pentaethylene glycol (476.56 mg, 0.423 ml, 2 mmol, 1 eq.)
was dissolved in 4 ml of dry DCM. The temperature of the resulting
mixture was cooled down to 0.degree. C. and methanesulfonyl
chloride (687.3 mg, 0.464 ml, 16 mmol, 3 eq.) was added followed by
triethylamine (1011.9 g, 1.39 ml, 10 mmol, 5 eq.). The resulting
mixture was stirred at 0.degree. C. for 4 h. A 10% aqueous solution
of NaHSO.sub.4 was added till pH=3. The aqueous phase was extracted
with DCM (.times.5). The organic phases were combined, dried over
MgSO.sub.4 and concentrated in vacuum to afford the title compound
as an orange oil. Yield: 701 mg, 1.77 mmol (89%).
[0212] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.33-4.30 (4H, m),
3.72-3.69 (4H, m), 3.62-3.56 (12H, m), 3.02 (6H, s). Analytical
data matched those previously reported [Kimura et al. J. Polym.
Sci. Part A: Polym. Chem. 54, (2016).]
tert-butyl
17-((methylsulfonyl)oxy)-3,6,9,12,15-pentaoxaheptadecanoate
(20)
##STR00016##
[0214] 10 (251 mg, 0.712 mmol, 1 eq.) was dissolved in 1.4 ml of
dry DCM. The temperature of the resulting mixture was cooled down
to 0.degree. C. and methanesulfonyl chloride (122.3 mg, 0.082 ml,
1.068 mmol, 1.5 eq.) was added followed by triethylamine (216.14
mg, 0.3 ml, 2.136 ml, 3 eq.). The resulting mixture was stirred at
0.degree. C. for 4 h. A 10% aqueous solution of NaHSO.sub.4 was
added till pH=3. The aqueous phase was extracted with DCM
(.times.5). The organic phases were combined, dried over MgSO.sub.4
and concentrated in vacuum to afford the title compound as a orange
oil. Yield: 276 mg, 0.641 mmol (90%).
[0215] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.32-4.30 (2H, m),
3.95 (2H, s), 3.71-3.57 (18H, m), 3.02 (3H, s), 1.41 (9H, s).
.sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 169.7, 81.5, 70.72,
70.65, 70.61, 70.58, 70.5, 69.3, 69.0, 37.7, 28.1.
tert-butyl
(S)-19-((S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl))benzyl)ca-
rbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-
-18-azahenicosanoate (12)
##STR00017##
[0217] To a solution of PEG linker 11 (78.8 mg , 0.215 mmol, 1 eq.)
in 1.5 ml DMF was added HATU (81.74 mg, 0.215 mmol, 1 eq.), HOAT
(29.26 mg, 0.215 mmol, 1 eq.), DIPEA and the solution was stirred
at room temperature for 5 min. Compound 7 (100 mg, 0.215 mmol, 1
eq.) was added and the pH of the reaction mixture was adjusted to
>9 by addition of DIPEA(80.13 mg, 0.106 ml, 0.645 mmol, 3 eq.).
The mixture was stirred at room temperature until no presence of
the starting materials was detected by LC-MS. The solvent was
evaporated under reduced pressure to give the corresponding crude,
which was purified by HPLC using a gradient of 20% to 95% v/v
acetonitrile in 0.1% aqueous solution of ammonia to yield the
titled compound as white solid. Yield: 75.6 mg, 0.094 mmol
(44%).
[0218] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. ppm 9.00 (1H, s),
7.45 (1H, t, J=5.9 Hz), 7.39-7.33 (4H, m), 7.29 (1H, d, J=8.9 Hz),
4.71 (1H, t, J=8.0 Hz), 4.59-4.48 (3H, m), 4.34 (1H, dd, J=5.2,
14.6 Hz), 4.08-3.92 (5H, m), 3.69-3.61 (18H, m), 2.52 (3H, s),
2.47-2.41 (1H, m), 2.19-2.11 (1H, m), 1.46 (9H, s), 0.97 (9H, s).
.sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 171.3, 171.1, 170.5,
170.0, 151.7, 139.1, 129.4, 128.3, 82.0, 71.1, 70.6, 70.4, 70.4,
70.3, 70.3, 70.2, 70.2, 68.9, 58.7, 57.3, 56.8, 43.1, 36.3, 35.1,
28.1, 26.4, 15.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for:
C.sub.38H.sub.58N.sub.4O.sub.11S.sub.2: 778.38; observed:
779.4.
[0219]
N.sup.1--((R)-1-((2R,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benz-
yl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-N.sup.17--((S)-
-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrroli-
din-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15-pentaoxaheptadecanedia-
mide (CMP99)
##STR00018##
[0220] Following general method B, from compound 12 (75.6 mg, 0.094
mmol, 1 eq.) and trifluoroacetic acid (1 ml in 1 ml of DCM), the
carboxylic acid derivative was obtained as oil. The crude was used
for the next step without further purification. Yield: 70 mg, 0.094
mmol (quantitative). MS (ESI) m/z: [M+H].sup.+ calculated for:
C.sub.34H.sub.50N.sub.4O.sub.11S: 722.32; observed: 723.3.
Following general method B, from compound 13 (5.5 mg, 0.006 mmol, 1
eq.), compound 8 (2.77 mg, 0.006 mmol, 1 eq.), HATU (2.28 mg,
0.0.006 mmol, 1 eq.), HOAT (1 mg, 0.0.006 mmol, 1 eq.), DIPEA (2.23
mg, 0.002 ml, 0.018 mmol, 3 eq.), CMP99 was obtained as a white
solid. Yield: 4.5 mg, 0.004 mmol (66%).
[0221] .sup.1H NMR (400 MHz, CDCl3): d, ppm 8.74 (2H, d, J=2.8 Hz),
7.37-7.34 (9H, m), 7.18 (1H, d, J=8.9 Hz), 4.76-4.64 (3H, m),
4.59-4.44 (5H, m), 4.37-4.26 (2H, m), 4.05-3.59 (27H, m), 2.52 (6H,
s), 2.31-2.11 (4H, m), 0.96 (9H, s), 0.95 (9H, s). HRMS (ESI) m/z:
[M+H].sup.+ calculated for: C.sub.56H.sub.78N.sub.8O.sub.13S.sub.2:
1134.51; observed: 1135.58.
N.sup.1,N.sup.14-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)-
benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12-t-
etraoxatetradecanediamide (CM09)
##STR00019##
[0223] Following general method B, from compound 1 (6.80 mg, 0.018
mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg,
0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol), DIPEA (6.98
mg, 0.054 mmol, 3 eq) compound CM09 was obtained as a white solid.
Yield: 8 mg, 0.007 mmol (40%).
[0224] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (2H, s),
7.48-7.45 (2H, m), 7.31-7.27 (8H, m), 7.23 (2H, d, J=10.2 Hz),
4.64-4.59 (2H, m), 4.52-4.46 (4H, m), 4.41-4.38 (2H, m), 4.31-4.25
(2H, m), 4.01-3.94 (4H, m), 3.82 (2H, d, J=15.7 Hz), 3.62-3.52
(12H, m), 2.45 (6H, s), 2.42-2.34 (2H, m), 2.12-2.06 (2H, m), 1.19
(2H, s), 0.89 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta.
170.2, 169.9, 169.6, 149.3, 147.5, 137.3, 130.6, 129.9, 128.4,
127.1, 69.9, 69.5, 69.3, 69.1, 57.6, 56.1, 55.9, 42.2, 35.5, 34.6,
25.4, 15.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for:
C.sub.54H.sub.74N.sub.8O.sub.12S.sub.2: 1090.49; observed:
1091.4.
N.sup.1,N.sup.17-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)-
benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,1-
5-pentaoxaheptadecanediamide (CM10)
##STR00020##
[0226] Following general method B, from compound 2 (7.60 mg, 0.018
mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg,
0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol), DIPEA (6.98
mg, 0.054 mmol, 3 eq) compound CM10 was obtained as a white solid.
Yield: 6 mg, 0.005 mmol (30%).
[0227] .sup.1H NMR (400 MHz, MeOD) .delta. 9.28 (2H, s), 7.43-7.36
(10H, m), 5.39 (2H, s), 4.77 (10H, s), 4.59 (2H, s), 4.50-4.43 (4H,
m), 4.42-4.38 (2H, m), 4.26 (2H, d, J=17.2 Hz), 3.96-3.92 (4H, m),
3.77 (2H, d, J=11.1 Hz), 3.73-3.68 (2H, m), 3.56 (16H, m),
3.22-3.20 (10H, m), 2.43 (6H, s), 2.16-2.14 (2H, m), 2.13 (2H, m),
2.02-1.95 (2H, m); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta.
173.1, 172.4, 170.7, 170.3, 153.3, 153.2, 144.5, 140.0, 134.0,
129.2, 129.0, 128.4, 128.3, 127.8, 70.9, 70.5, 70.2, 70.1, 69.7,
68.2, 67.7, 59.7, 59.4, 56.8, 56.7, 54.9, 42.9, 42.3, 39.9, 37.6,
36.3, 35.7, 34.7, 25.6, 25.5, 13.1. HRMS (ESI) m/z: [M+H].sup.+
calculated for: C.sub.56H.sub.78N.sub.8O.sub.13S.sub.2: 1134.51;
observed: 1135.55.
N.sup.1,N.sup.20-bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)-
benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,1-
5,18-hexaoxaicosanediamide (CM11)
##STR00021##
[0229] Following general method B, from compound 3 (8.39 mg, 0.018
mmol, 1 eq.), compound 7 (20 mg, 0.045 mmol, 2.5 eq.), HATU (17 mg,
0.045 mmol, 2.5 eq), HOAT (6.12, 0.045 mmol, 2.5 mmol), DIPEA (6.98
mg, 0.054 mmol, 3 eq) compound CM11 was obtained as a white solid.
Yield: 11.74 mg, 0.0099 mmol (55%).
[0230] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (2H, s),
7.41-7.38 (2H, m), 7.29 (10H, t, J=7.6 Hz), 4.66-4.61 (2H, m),
4.49-4.41 (6H, m), 4.35-4.29 (2H, m), 3.98-3.91 (6H, m), 3.62-3.50
(24H, m), 2.45 (6H, s), 2.42-2.35 (2H, m), 2.11-2.06 (2H, m), 0.88
(18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 171.2, 170.9,
170.4, 150.3, 148.5, 138.3, 131.6, 130.9, 129.5, 128.1, 71.2,
70.61, 70.59, 70.5, 70.4, 70.3, 58.6, 57.0, 43.2, 36.5, 35.6, 26.4,
16.1. HRMS (ESI) m/z: [M+H].sup.+ calculated for:
C.sub.58H.sub.82N.sub.8O.sub.14S.sub.2: 1178.54; observed:
1179.60.
N.sup.1,N.sup.20-bis((S)-1-((2S,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)-
benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,1-
5,18-hexaoxaicosanediamide (CMP98)
##STR00022##
[0232] Following general method B, from compound 3 (7.12 mg, 0.028
mmol, 1 eq.), compound 8 (18.06, 0.040 mmol, 2.1 eq.), HATU (15.2
mg, 0.040 mmol, 2 eq.), HOAT (5.44 mg, 0.040 mmol, 2 eq.), DIPEA
(7.45 mg, 0.0010 ml, 3 eq.), compound CMP98 was obtained as a white
solid. Yield: 10.58 mg, 0.0089 mmol (45%).
[0233] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.09 (2H, s), 8.02
(2H, s), 7.31 (4H, d, J=8.5 Hz), 7.22 (4H, d, J=8.0 Hz), 7.16 (2H,
d, J=9.2 Hz), 4.75-4.64 (4H, m), 4.51 (2H, d, J=8.9 Hz), 4.41-4.37
(2H, m), 4.24-4.17 (2H, m), 3.94 (4H, d, J=3.2 Hz), 3.84-3.81 (4H,
m), 3.62-3.54 (20H, m), 2.49-2.47 (2H, m), 2.44 (6H, s), 2.26-2.17
(4H, m), 0.93 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta.
173.2, 171.5, 169.7, 151.8, 138.8, 132.9, 129.5, 129.2, 128.3,
71.2, 71.1, 70.6, 70.48, 70.45, 70.4, 70.3, 59.9, 58.5, 56.5, 43.2,
35.6, 35.2, 26.4, 15.0. HRMS (ESI) m/z: [M+H].sup.+ calculated for:
C.sub.58H.sub.82N.sub.8O.sub.14S.sub.2: 1178.54; observed:
1179.60.
(2S,2'S,4R,4'R)--N,N'-((((3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(oxy))-
bis(4-(4-methylthiazol-5-yl)-2,1-phenylene))bis(methylene))bis(1-((S)-2-(1-
-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidi-
ne-2-carboxamide) (CMP108)
##STR00023##
[0235] Following general method C, from 18 (27 mg, 0.05 mmol, 2
eq.), 19 (11.83 mg, 0.03 mmol, 1.2 eq.) and K.sub.2CO.sub.3 (41.46
mg, 0.3 mmol, 6 eq.), the titled compound was obtained as a white
solid. Yield: 9.1 mg, 0.007 mmol (28%).
[0236] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (2H, s),
7.41-7.38 (2H, m), 7.26 (2H, d, J=8.1 Hz), 7.00 (2H, d, J=8.1 Hz),
6.91-6.88 (2H, m), 6.85-6.81 (2H, m), 4.57-4.52 (2H, m), 4.44-4.36
(8H, m), 4.19-4.08 (4H, m), 3.89-3.53 (22H, m), 2.45 (6H, s),
2.24-2.17 (2H, m), 2.08-2.02 (2H, m), 1.61-1.37 (8H, m), 0.88 (18H,
s); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 170.9, 170.0, 165.4,
156.9, 150.4, 148.5, 132.3, 131.7, 130.0, 126.9, 122.0, 119.6,
112.9, 70.7, 70.41, 70.38, 70.2, 69.6, 67.9, 58.9, 58.4, 56.6,
39.2, 37.0, 36.0, 26.3, 17.9, 17.7, 16.2, 13.7. HRMS (ESI) m/z:
[M+H].sup.+ calculated for:
C.sub.64H.sub.84N.sub.10O.sub.14S.sub.2: 1280.56; observed:
1281.66.
(2S,2'S,4R,4'R)--N,N'-((((3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(oxy))-
bis(4-(4-methylthiazol-5-yl)-2,1-phenylene))bis(methylene))bis(1-((S)-2-ac-
etamido-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide)
(CMP106)
##STR00024##
[0238] Following general method C, from 17 (24.3 mg, 0.05 mmol, 2
eq.), 19 (11.83 mg, 0.03 mmol, 1.2 eq.) and and K.sub.2CO.sub.3
(41.46 mg, 0.3 mmol, 6 eq.), the title compound was obtained as a
white solid. Yield: 7.8 mg, 0.006 mmol (26%).
[0239] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.60 (2H, s),
7.39-7.35 (2H, m), 7.26 (2H, d, J=7.6 Hz), 6.91-6.88 (2H, m),
6.83-6.80 (2H, m), 6.36-6.13 (2H, m), 4.60-4.32 (10H, m), 4.18-4.05
(4H, m), 3.97-3.79 (6H, m), 3.71-3.54 (18H, m), 2.44 (6H, s),
2.17-1.86 (8H, m), 0.87 (18H, s); .sup.13C NMR (101 MHz,
CDCl.sub.3) .delta. 171.3, 171.1, 171.0, 170.7, 170.5, 156.8,
156.8, 150.3, 148.5, 132.2, 131.7, 130.0, 129.8, 127.1, 126.9,
122.1, 122.0, 112.8, 112.8, 71.3, 70.7, 70.6, 70.5, 70.5, 70.5,
70.4 , 70.2, 70.1, 69.7, 67.9, 58.9, 58.6, 57.6, 57.5, 56.9, 56.7,
42.7, 39.1, 39.0, 37.1, 36.4, 35.4, 35.1, 26.4, 26.4, 23.2, 23.1,
16.2. HRMS (ESI) m/z: [M+H].sup.+ calculated for:
C.sub.58H.sub.82N.sub.8O.sub.14S.sub.2: 1178.54; observed:
1281.66.
tert-butyl(14-(2-(((2S,4R)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,-
3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methyl-
thiazol-5-yl)phenoxy)-3,6,9,12-tetraoxatetradecyl) carbonate
(22)
##STR00025##
[0241] Following general method C, from 18 (27 mg, 0.05 mmol, 1
eq.), 20 (26 mg, 0.06 mmol, 1.2 eq.) and K.sub.2CO.sub.3 (20.73 mg,
0.15 mmol, 3 eq.), the title compound was obtained as a white
solid. Yield: 17 mg, 0.02 mmol (33%).
[0242] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (1H, s),
7.33-7.25 (2H, m), 6.97 (1H, d, J=9.1 Hz), 6.92-6.89 (1H, m), 6.84
(1H, d, J=1.5 Hz), 4.59-4.55 (1H, m), 4.45-4.38 (4H, m), 4.22-4.10
(2H, m), 3.93-3.54 (24H, m), 2.46 (3H, s), 2.32-2.24 (1H, m),
2.10-2.04 (1H, m), 1.63-1.52 (2H, m), 1.45-1.39 (12H, m), 0.87 (9H,
s); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 170.6, 170.1, 169.7,
165.4, 156.9, 150.3, 148.5, 132.3, 131.7, 130.0, 126.9, 122.0,
119.7, 112.9, 81.7, 70.72, 70.66, 70.5, 70.4, 70.3, 69.6, 69.0,
68.0, 58.8, 58.4, 56.6, 39.3, 36.7, 35.8, 28.1, 26.3, 17.8, 16.2,
13.7. HRMS (ESI) m/z: [M+H].sup.+ calculated for:
C.sub.43H.sub.63N.sub.5O.sub.12S: 873.42; observed: 874.49.
tert-butyl
17-(2-(((2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydr-
oxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-3,6,-
9,12,15-pentaoxaheptadecanoate (21)
##STR00026##
[0244] Following general method C, from 17 (24.3 mg, 0.05 mmol, 1
eq.), 20 (26 mg, 0.06 mmol, 1.2 eq.) and K.sub.2CO.sub.3 (20.73 mg,
0.15 mmol, 3 eq.), the title compound was obtained as a white
solid. Yield: 17 mg, 0.021 mmol (33%).
[0245] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta., ppm 8.67 (1H, s),
7.32 (2H, d, J=7.8 Hz), 6.95 (1H, dd, J=1.6, 7.6 Hz), 6.88 (1H, d,
J=1.8 Hz), 4.65-4.60 (1H, m), 4.53-4.43 (2H, m), 4.39-4.36 (1H, m),
4.24-4.13 (2H, m), 4.00 (2H, d, J=7.0 Hz), 3.92-3.87 (2H, m),
3.77-3.59 (20H, m), 3.08 (2H, s), 2.51 (3H, s), 2.38-2.31 (1H, m),
1.98 (3H, s). .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 171.2,
170.8, 170.4, 169.7, 156.8, 150.3, 148.5, 132.2, 131.7, 129.8,
126.9, 122.0, 112.8, 81.6, 70.8, 70.71, 70.69, 70.60, 70.57, 70.55,
70.52, 70.49, 70.47, 70.1, 69.6, 69.3, 69.02, 68.98, 67.9, 58.6,
57.5, 56.7, 39.0, 37.7, 36.5, 35.2, 28.1, 26.4, 23.2, 16.1. HRMS
(ESI) m/z: [M+H].sup.+ calculated for:
C.sub.40H.sub.62N.sub.4O.sub.12S: 822.41; observed: 823.5.
(2S,4R)-1-((S)-2-(tert-butyl)-20-(2-(((2S,4R)-1-((S)-2-(1-cyanocyclopropan-
e-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-
methyl)-5-(4-methylthiazol-5-yl)phenoxy)-4-oxo-6,9,12,15,18-pentaoxa-3-aza-
icosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carbo-
xamide (CMP113)
##STR00027##
[0247] Following general method B, from compound 20 (17 mg, 0.02
mmol, 1 eq.) and trifluoroacetic acid (0.5 ml in 0.5 ml of DCM),
the carboxylic acid derivative was obtained as an oil. Yield: 15.7
mg, 0.019 mmol (quantitative). HRMS (ESI) m/z: [M+H].sup.+
calculated for: C.sub.39H.sub.55N.sub.5O.sub.12S: 817.36; observed:
818.4.
[0248] From the obtained carboxylic acid (15.7 mg, 0.019 mmol, 1
eq.) in 0.5 ml DMF, HATU (7.22 mg, 0.019 mmol, 1 eq.), HOAT (2.58
mg, 0.019 mmol, 1 eq.), compound 7 (8.73 mg, 0.019 mmol, 1 eq.) and
DIPEA (3 eq.), the final compound was isolated as white solid.
Yield: 6.3 mg, 0.005 mmol (27%).
[0249] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (2H, s),
7.58-7.54 (1H, m), 7.31-7.25 (5H, m), 7.02 (1H, d, J=9.7 Hz),
6.88-6.85 (1H, m), 6.78 (1H, d, J=1.5 Hz), 4.59-4.56 (2H, m),
4.47-4.25 (6H, m), 4.13-3.52 (20H, m), 2.47-2.42 (6H, m), 2.34-2.27
(1H, m), 2.19-2.03 (5H, m), 1.63-1.52 (2H, m), 1.48-1.37 (2H, m),
0.90 (18H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 171.2,
171.1, 170.7, 170.3, 165.4, 156.6, 150.3, 148.4, 148.3, 138.3,
132.0, 131.8, 131.7, 130.7, 129.5, 129.4, 127.9, 126.9, 122.0,
119.7, 112.6, 71.0, 70.7, 70.5, 70.4, 70.32, 70.28, 70.25, 69.6,
67.9, 59.1, 58.8, 58.5, 57.3, 57.1, 56.7, 43.1, 39.0, 37.3, 36.8,
36.2, 35.4, 26.4, 26.3, 17.9, 17.7, 16.1, 16.0, 13.7. HRMS (ESI)
m/z: [M+H].sup.+ calculated for:
C.sub.61H.sub.83N.sub.9O.sub.14S.sub.2: 1229.55; observed:
1230.66.
(2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-((S)-19-((-
2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine--
1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosyl)oxy-
)-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(CMP112)
##STR00028##
[0251] Following general method B, from compound 20 (17 mg, 0.021
mmol, 1 eq.) and trifluoroacetic acid (0.5 ml in 0.5 ml of DCM),
the carboxylic acid derivative or 38 was obtained as an oil. Yield:
13 mg, 0.017 mmol (quantitative). HRMS (ESI) m/z: [M+H].sup.+
calculated for: C.sub.36H.sub.54N.sub.4O.sub.12S: 766.35; observed:
767.4.
[0252] From the carboxylic acid (13 mg, 0.017 mmol, 1 eq.) in 0.5
ml DMF, HATU (6.49 mg, 0.017 mmol, 1 eq.), HOAT (2.31 mg, 0.017
mmol, 1 eq.), compound 7 (7.90 mg, 0.017 mmol, 1 eq.) and DIPEA (3
eq.)the titled compound was obtained as a white solid. Yield: 6 mg,
0.005 mmol (30%).
[0253] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (2H, s),
7.49-7.45 (1H, m), 7.32-7.24 (6H, m), 6.90-6.87 (1H, m), 6.79 (1H,
d, J=2.4 Hz), 6.24 (1H, d, J=8.9 Hz), 4.61-4.29 (10H, m), 4.11-3.52
(27H, m), 2.44 (6H, s), 2.30 (1H, t, J=13.3 Hz), 2.18-2.03 (3H, m),
0.87 (9H, s); .sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 171.2,
171.1, 170.7, 170.4, 156.7, 150.3, 148.4, 138.3, 132.2, 130.9,
129.7, 129.4, 128.0, 127.0, 122.0, 112.8, 70.9, 70.6, 70.5, 70.4,
70.3, 70.2, 69.6, 67.9, 59.0, 58.8, 57.7, 57.1, 43.1, 39.0, 37.1,
36.8, 35.6, 35.5, 26.42, 26.38, 23.0, 16.13, 16.06. HRMS (ESI) m/z:
[M+H].sup.+ calculated for: C.sub.58H.sub.82N.sub.8O.sub.14S.sub.2:
1178.54; observed: 1179.6.
[0254] General Method D:
[0255] To a solution of the diol (1 eq.) in DCM, tert-butyl
bromoacetate (8 eq.), TBABr (1.1 eq.) and 37% w/w aqueous NaOH were
added. The reaction mixture was vigorously stirred at r.t.
overnight. The organic phase was separated from the aqueous layer
and then the aqueous phase was extracted with DCM (.times.3).
Organic layers were collected, dried over MgSO.sub.4 and evaporated
under reduced pressure. The crude was purified by flash
chromatography eluting with ethyl acetate from 10% to 50% v/v in
heptane.
[0256] General Method E:
[0257] A solution of the benzylated starting material in absolute
EtOH (0.05 M) was flown in an H-cube machine at a rate of 1 mL/min,
H.sub.2 10 atm, 70.degree. C. Solvent was evaporated under reduced
pressure to yield the final compound.
[0258] General Method F:
[0259] To a solution of the dicarboxylic acid linker (1 eq.) in dry
DMF, COMU (2 eq.) and DIPEA (5 eq.) were added. The solution was
stirred for 10 min and then it was added to a suspension of the
VHL-ligand amine 7 (2.1 eq.) and DIPEA (5 eq.) in dry DMF. The
mixture was stirred at r.t. until no presence of the starting
material was detected by LC-MS. Ice was added and the volatiles
were evaporated under reduced pressure to give the crude which was
purified by HPLC using a gradient of 20% to 70% v/v acetonitrile in
0.1% v/v aqueous solution of formic acid to yield the final
compound.
[0260] 4,4'-(Butane-1,4-diylbis(oxy))bis(butan-1-ol) (101)
##STR00029##
[0261] Compound 101 was prepared as reported.sup.40 by Knuf et al.
Analytical data matched those previously reported.
Di-tert-butyl 3,8,13,18-tetraoxaicosanedioate (102)
##STR00030##
[0263] Prepared following the general method D from compound 101
(198 mg, 0.8449 mmol) in 37% w/w aqueous NaOH (4 mL) and DCM (4
mL). Compound 102 was obtained as an oil (158 mg, yield: 40%).
[0264] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 3.87(4H, s), 3.45
(4H, t, J=6.1 Hz), 3.38-3.30 (8H, m), 1.67-1.51 (12H, m), 1.41
(18H, s).
3,8,13,18-Tetraoxaicosanedioic acid (103)
##STR00031##
[0266] Prepared following the general method B starting from
compound 102 (158 mg, 0.3415 mmol) in TFA/DCM 1:1 (2 mL). Compound
103 was obtained as an oil (120 mg, yield: quantitative).
[0267] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.26 (s, 2H),
4.09 (s, 4H), 3.58 (t, J=6.1 Hz, 4H), 3.48-3.41 (m, 8H), 1.75-1.60
(m, 12H)..sup.13C-NMR (101 MHz, CDCl.sub.3) .delta.: 173.1, 71.7,
70.6, 70.4, 67.9, 26.4, 26.3, 26.1.
5-(Benzyloxy)pentan-1-ol (104)
##STR00032##
[0269] 1,5-Pentandiol (3.430 g, 3.45 mL, 0.033 mol, 4 eq.) was
added dropwise to a suspension of NaH (670 mg, 0.016 mol, 2 eq) in
DMF (14 mL) at 0.degree. C. A catalytic amount of Nal was added,
followed by benzylbromide (1.360 g, 0.95 mL, 0.008 mol, 1 eq.). The
mixture was stirred at r.t. overnight.
[0270] The reaction was quenched with NH.sub.4Cl aq. sat. and then
extracted with ethyl acetate (.times.3).
[0271] Organic layers were collected and evaporated under reduced
pressure. The crude was purified by flash chromatography eluting
from 40% to 90% of ethyl acetate in heptane to give the desired
product (1.08 g, yield: 70%).
[0272] Analytical data matched those previously
reported..sup.41
2-(2-(2-(Benzyloxy)ethoxy)ethoxy)ethyl methanesulfonate (105)
##STR00033##
[0274] Compound 105 was obtained following the method previously
reported.sup..42 Analytical data matched those reported.
1,18-Diphenyl-2,5,8,11,17-pentaoxaoctadecane (106)
##STR00034##
[0276] Compound 104 (228.58 mg, 1.177 mmol, 2.5 eq) was added to a
solution of NaHMDS 1M in THF (107.95 mg, 0.588 mL, 0.588 mmol, 1.25
eq.) at 0.degree. C. under N.sub.2 atmosphere. Reaction mixture was
stirred at r.t. for 1 h. After this time a solution of compound 105
(150.00 mg, 0.471 mmol, 1 eq.) in DMF was added and the mixture was
irradiated with microwave at 130.degree. C. for 2 h.
[0277] After this time the solvent was evaporated, the reaction
quenched with 5% aqueous NaHSO.sub.4 and extracted with DCM
(.times.3). Organic layers were collected, dried over MgSO.sub.4,
filtered and evaporated under reduced pressure. The crude was
purified by flash chromatography eluting from 0% to 50% v/v of
ethyl acetate in heptane to yield the desired compound 106 as an
oil (114 mg, yield: 58%).
[0278] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 7.28-7.19 (m,
10H), 4.49 (s, 2H), 4.43 (s, 2H), 3.62-3.54 (m, 10H), 3.51-3.48 (m,
2H), 3.43-3.36 (m, 4H), 1.61-1.48 (m, 4H), 1.42-1.31 (m, 2H).
Di-tert-butyl 3,6,9,12,18-pentaoxaicosanedioate (107)
##STR00035##
[0280] Starting from compound 106 (265 mg, 0.610 mmol) and
following the general method E the deprotected compound was
obtained as an oil (131 mg) and used without any further
purification for the next step.
[0281] Following the general method D from the deprotected compound
(131 mg, 0.5544 mmol) in 37% w/w aqueous NaOH (2.2 mL) and DCM (2.2
mL) compound 107 was obtained as an oil (122 mg, yield: 47%).
[0282] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 4.00 (s, 2H),
3.92 (s, 2H), 3.69-3.60 (m, 10H), 3.57-3.53 (m, 2H), 3.52-3.46 (m,
2H), 3.43 (t, J=7.1 Hz, 2H), 1.67-1.56 (m, 4H), 1.46 (d, J=0.6 Hz,
18H), 1.43-1.37 (m, 2H). .sup.13C-NMR (101 MHz, CDCl.sub.3)
.delta.: 169.8, 81.5, 81.4, 71.6, 71.3, 70.7, 70.6, 70.1, 69.0,
68.8, 29.5, 29.4, 28.1, 22.6.
3,6,9,12,18-Pentaoxaicosanedioic acid (108)
##STR00036##
[0284] Prepared following the general method B starting from
compound 107 (90 mg, 0.1937 mmol) in 2 mL of TFA/DCM 1:1. Compound
108 was obtained as an oil (66 mg, yield: quantitative).
[0285] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.15 (s, 2H),
4.11 (s, 2H), 4.02 (s, 2H), 3.71-3.40 (m, 16H), 1.65-1.52 (m, 4H),
1.43-1.34 (m, 2H)
1,5-Bis(allyloxy)pentane (109)
##STR00037##
[0287] Compound 109 was obtained starting from 1,5-petandiol (500
mg, 4.8 mmol) and following the method reported..sup.43
[0288] Analytical data matched those previously reported.
3,3'-(Pentane-1,5-diylbis(oxy))bis(propan-1-ol) (110)
##STR00038##
[0290] A solution of compound 109 (500 mg, 2.71 mmol, 1 eq.) in dry
THF (4.2 mL) was added dropwise to a solution 0.5 M of
9-Borabicyclo[3.3.1]nonane in THF (993 mg, 16.28 mL, 8.14 mmol,
3eq.) at 0.degree. C. and the resulting solution was stirred at
r.t. overnight.
[0291] The reaction was quenched by MeOH (3.17 mL), 30% w/w aq.
NaOH (6.35 mL), 30% v/v aq. H.sub.2O.sub.2 (6.35 mL) and the
mixture was left to stir for 2 h. Then it was extracted with ethyl
acetate (.times.3). Organic layers were collected, washed with
brine, dried over MgSO.sub.4 and evaporated under reduced pressure.
The crude was purified by flash chromatography eluting from 0% to
100% ethyl acetate in heptane to yield the desired product as an
oil (483 mg, yield: 81%). Analytical data matched those previously
reported..sup.43
Di-tert-butyl 3,7,13,17-tetraoxanonadecanedioate (111)
##STR00039##
[0293] Compound 111 was obtained from compound 110 (214 mg, 0.9714
mmol) following the general method D, in 37% w/w aqueous NaOH (4
mL) and DCM (4 mL). The desired product was obtained as an oil (65
mg, yield: 15%).
[0294] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 3.88 (s, 4H),
3.53 (t, J=6.5 Hz, 4H), 3.44 (t, J=6.4 Hz, 4H), 3.34 (t, J=6.9 Hz,
4H), 1.85-1.78 (m, 4H), 1.55-1.47 (m, 4H), 1.41 (s, 18H), 1.36-1.29
(m, 2H).
3,7,13,17-Tetraoxanonadecanedioic acid (112)
##STR00040##
[0296] Prepared following the general method B starting from
compound 111 (64 mg, 0.1427 mmol) in TFA/DCM 1:1 (2 mL). Compound
112 was obtained as an oil (47.5 mg, yield: quantitative).
[0297] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 8.11 (s, 2H),
4.06 (s, 4H), 3.64 (t, J=5.9 Hz, 4H), 3.54 (t, J=5.9 Hz, 4H), 3.42
(t, J=6.4 Hz, 4H), 1.88-1.81 (m, 4H), 1.60-1.52 (m, 4H), 1.36 (dt,
J=7.6, 11.9 Hz, 2H). .sup.13C-NMR (101 MHz, CDCl.sub.3) .delta.:
173.3, 71.1, 69.6, 68.2, 67.9, 29.4, 29.2, 22.7.
5-(Benzyloxy)pentyl 4-methylbenzenesulfonate (113)
##STR00041##
[0299] To a solution of compound 104 (1.910 g, 9.8387 mmol, 1 eq.)
and triethylamine (1.65 mL, 11.8226 mmol, 1.2 eq.) in DCM (15 mL) a
solution of p-TsCl (2.063 g, 10.8226 mmol, 1.1 eq.) in DCM (15 mL)
was added at 0.degree. C. The mixture was left to stir overnight.
Then NaHCO.sub.3 aq. sat. was added. The aqueous phase was
separated from the organic layer and it was extracted with DCM
(.times.2). Organic layers were collected and washed with 5%
aqueous HCl solution. The crude was purified by flash
chromatography eluting from 0% to 60% v/v ethyl acetate in heptane
to yield the desired product (1.9 g, yield: 55%). Analytical data
matched those previously reported..sup.44
1,18-Diphenyl-2,8,11,17-tetraoxaoctadecane (114)
##STR00042##
[0301] A mixture of compound 113 (1.9 g, 5.6863 mmol, 2.4 eq.),
ethylenglycol (147 mg, 2.3696 mmol, 1 eq.) and TBA bisulphate (804
mg, 2.3693 mmol, 1 eq) was dissolved in toluene (8 mL) and NaOH aq.
50% (6 mL). The mixture was vigorously stirred overnight. The
organic phase was separated from the aqueous layer and then it was
extracted with ethyl acetate (.times.3). Organic layers were
collected, dried over MgSO.sub.4 and evaporated under reduced
pressure. The crude was purified by flash chromatography eluting
with a mixture v/v of ethyl acetate in heptane, from 100% heptane
to 100% ethyl acetate. The desired compound was obtained as an oil
(200 mg, yield: 8.5%).
[0302] .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 7.25-7.22 (m,
10H), 4.40 (s, 4H), 3.47 (s, 4H), 3.39-3.35 (m, 8H), 1.58-1.47 (m,
8H), 1.37-1.29 (m, 4H).
5,5'-(Ethane-1,2-diylbis(oxy))bis(pentan-1-ol (115)
##STR00043##
[0304] Starting from compound 114 (200 mg, 0.8535 mmol) and
following the general method E compound 115 was obtained as an oil
(35 mg, yield: 31%).
[0305] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 3.53 (t, J=6.1
Hz, 4H), 3.49 (s,4H), 3.49 (s, 4H), 3.40 (t, J=6.6 Hz,4H), 2.93(s,
2H), 1.58-1.45(m, 8H), 1.37-1.29 (m, 4H).
1,2-Di(1,3-dioxan-2-yl)ethane (118)
##STR00044##
[0307] Compound 118 was prepared in accordance with the published
procedure,.sup.45 starting from 2,5-dimethoxytetrahydrofuran (10.0
g, 75.6659 mmol). Analytical data matched those previously
reported.
3,3'-(Butane-1,4-diylbis(oxy))bis(propan-1-ol) (119)
##STR00045##
[0309] Compound 119 was prepared accordingly in accordance with the
published procedure,.sup.45 starting from compound 118. Analytical
data matched those previously reported.
1-Phenyl-2,5,9,14-tetraoxaheptadecan-17-ol (120)
##STR00046##
[0311] Compound 120 (1.1 g, 5.3325 mmol, 3 eq.) was dissolved in
toluene (10 mL) and NaOH aq. 50% w/w (5 mL). TBABr (590 mg, 1.7775
mmol, 1 eq.), a catalytic amount of TBAI and benzyl-2-bromoethyl
ether (382 mg, 1.7775 mmol, 1 eq.) were added and the reaction
mixture was vigorously stirred for 48 h. Organic layer was
separated from the aqueous phase and the aqueous phase was
extracted with DCM (.times.3). The crude was purified by flash
chromatography eluting from 0% to 5% v/v MeOH in DCM to obtain the
product as an oil (350 mg, 57%).
[0312] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 7.33-7.22 (m,
5H), 4.55 (s, 2H), 3.74 (dd, J=5.7, 11.2 Hz, 2H), 3.59-3.55 (m,
6H), 3.53 (t, J=6.5 Hz, 2H), 3.47 (t, J=6.4 Hz, 2H), 3.44-3.37 (m,
4H), 2.44 (t, J=5.7 Hz, 1H), 1.87-1.76 (m, 4H), 1.61-1.57 (m,
4H).
3-(4-(3-(2-Hydroxyethoxy)propoxy)butoxy)propan-1-ol (121)
##STR00047##
[0314] The product was obtained starting from compound 120 (350 mg,
1.028 mmol) and following the general method E. The conversion was
not quantitative so the product 121 was separated from the starting
material by a flash chromatography eluting from 100% DCM to 9:1 v/v
DCM/MEOH (87 mg, yield: 34%).
[0315] .sup.1H-NMR (400 MHz, CDCl3) .delta.: 3.66-3.60 (m, 4H),
3.51-3.38 (m, 8H), 3.38-3.31 (m, 4H), 1.79-1.69 (m, 4H), 1.57-1.50
(m, 4H).
Di-tert-butyl 3,6,10,15,19-pentaoxahenicosanedioate (122)
##STR00048##
[0317] Compound 122 was obtained from compound 121 (87 mg, 0.3475
mmol) following the general method D, in 37% w/w aqueous NaOH (1.5
mL) and DCM (1.5 mL). The desired product was obtained as an oil
(47 mg, yield: 28%).
[0318] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.: 3.99 (s, 2H),
3.68-3.42 (m, 12H), 3.41-3.36 (m, 4H), 1.88-1.77 (m, 4H), 1.59-1.55
(m, 4H), 1.44 (s, 18H).
3,6,10,15,19-Pentaoxahenicosanedioic acid (123)
##STR00049##
[0320] Prepared following the general method B starting from
compound 122 (47 mg, 0.0983 mmol) in TFA/DCM 1:1 (1 mL). Compound
123 was obtained as an oil (35 mg, yield: quantitative).
.sup.1H-NMR (500 MHz, CDCl3) .delta.: 4.14 (s, 2H), 4.07 (s, 2H),
3.73-3.69 (m, 2H), 3.65-3.59 (m, 4H), 3.59-3.53 (m, 4H), 3.49 (t,
J=6.3 Hz, 2H), 3.47-3.40 (m, 4H), 1.89-1.81 (m, 4H), 1.62-1.57 (m,
4H).
[0321] 13C-NMR (101 MHz, CDCl3) .delta.: 173.9, 173.7, 71.3, 71.0,
70.8, 70.0, 69.6, 68.7, 68.6, 68.1, 68.0, 67.6, 29.7, 29.5, 26.3,
26.2.
[0322]
N1,N20-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)ben-
zyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,18-p-
entaoxaicosanediamide (124)
##STR00050##
[0323] Compound 124 was prepared accordingly to general method F,
starting from compound 7 (20 mg, 0.0428 mmol) and compound 108 (7.2
mg, 0.02038 mmol). 5 mg were obtained (yield: 21%).
[0324] .sup.1H-NMR (500 MHz, MeOD) .delta.: 8.77 (s, 2H), 7.34 (dd,
J=7.4, 23.2 Hz, 8H), 4.59 (dd, J=2.4, 9.4 Hz, 2H), 4.50-4.38 (m,
6H), 4.27 (t, J=4.3 Hz, 1H), 4.24 (t, J=4.3 Hz, 1H), 3.94 (dd,
J=15.3, 22.3 Hz, 2H), 3.85 (dd, J=15.3, 24.4 Hz, 2H), 3.76 (d,
J=10.7 Hz, 2H), 3.72-3.68 (m, 2H), 3.61-3.40 (m, 14H), 3.35 (dt,
J=1.0, 6.5 Hz, 2H), 2.37 (s, 6H), 2.16-2.09 (m, 2H), 2.02-1.96 (m,
2H), 1.57-1.45 (m, 4H), 1.39-1.32 (m, 2H), 0.94 (s, 18H).
[0325] .sup.13C-NMR (101 MHz, MeOD) .delta.: 174.4, 174.3, 172.1,
172.0, 171.7, 152.9, 149.0, 140.3, 133.4, 131.5, 130.5, 130.4,
129.5, 129.0, 72.9, 72.3, 72.2, 71.7, 71.6, 71.5, 71.2, 71.1, 70.7,
60.8, 58.1, 58.0, 43.7, 38.9, 37.2, 37.1, 30.5, 30.4, 27.0, 26.9,
23.8, 15.8.
[0326] HRMS: found 1177.6435 [M+H.sup.+].
[0327]
N1,N20-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)ben-
zyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,8,13,18-tet-
raoxaicosanediamide (125)
##STR00051##
[0328] Compound 125 was prepared accordingly to general method F,
starting from compound 7 (20 mg, 0.0428 mmol) and compound 103 (7.1
mg, 0.02038 mmol). 6.7 mg were obtained (yield: 28%).
[0329] .sup.1H-NMR (500 MHz, MeOD) .delta.: 8.77 (s, 2H), 7.34 (dd,
J=8.3, 23.6 Hz, 8H), 4.59 (d, J=12.0 Hz, 2H), 4.50-4.40 (m, 6H),
4.26 (dd, J=4.9, 15.9 Hz, 2H), 3.86 (dd, J=15.3, 23.4 Hz, 4H), 3.77
(d, J=11.4 Hz, 2H), 3.70 (dd, J=3.9, 11.1 Hz, 2H), 3.46 (t, J=6.0
Hz, 4H), 3.38-3.28 (m, 8H), 2.37 (s, 6H), 2.16-2.10 (m, 2H),
2.02-1.96 (m, 2H), 1.62-1.52 (m, 8H), 1.51-1.45 (m, 4H), 0.93 (s,
18H).
[0330] .sup.13C-NMR (101 MHz, MeOD) .delta.: 174.3, 172.1, 172.0,
171.7, 152.8, 149.1, 140.3, 133.4, 131.5, 130.5, 130.4, 129.5,
129.0, 72.7, 71.7, 71.5, 71.1, 70.7, 60.8, 58.1, 58.0, 43.7, 39.0,
37.2, 27.6, 27.5, 27.4, 15.9.
[0331] HRMS: found 1175.6623 [M+H.sup.+].
N1,N19-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)car-
bamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,7,13,17-tetraoxano-
nadecanediamide (126)
##STR00052##
[0333] Compound 126 was prepared accordingly to general method F,
starting from compound 7 (20 mg, 0.0428 mmol) and compound 112 (6.8
mg, 0.0204 mmol). 6.6 mg were obtained as a white solid (yield:
28%).
[0334] .sup.1H-NMR (500 MHz, MeOD) .delta.: 8.76 (s, 2H), 7.37-7.30
(m, 8H), 4.60 (d, J=9.4 Hz, 2H), 4.50-4.24 (m, 8H), 3.87 (d, J=6.5
Hz, 4H), 3.77 (d, J=11.2 Hz, 2H), 3.70 (dd, J=3.8, 11.5 Hz, 2H),
3.55-3.49 (m, 4H), 3.43 (dt, J=1.2, 6.2 Hz, 4H), 3.33-3.29 (m, 4H),
2.37 (s, 6H), 2.16-2.10 (m, 2H), 2.03-1.96 (m, 2H), 1.80-1.74 (m,
4H), 1.47-1.40 (m, 4H), 1.30-1.23 (m, 2H), 0.93 (s, 18H).
[0335] .sup.13C-NMR (101 MHz, MeOD) .delta.: 174.3, 171.8, 171.6,
152.8, 149.0, 140.2, 133.4, 131.5, 130.5, 130.3, 129.5, 128.9,
71.9, 71.0, 70.8, 69.8, 68.3, 60.8, 58.1, 57.9, 43.7, 38.9, 37.2,
30.9, 30.5, 26.9, 23.9, 15.8.
[0336] HRMS: found 1161.6446 [M+H.sup.+].
N1,N21-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)car-
bamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,10,15,19-pentaox-
ahenicosanediamide (128)
##STR00053##
[0338] Compound 128 was prepared accordingly to general method F,
starting from compound 7 (20 mg, 0.0428 mmol) and compound 123 (7.5
mg, 0.02038 mmol). 6.5 mg were obtained as a white solid (yield:
27%).
[0339] .sup.1H-NMR (500 MHz, MeOD) .delta.: 9.00 (d, J=1.1 Hz, 2H),
7.45 (dd, J=8.4, 23.1 Hz, 8H), 4.71-4.68 (m, 2H), 4.55 (tt, J=12.4,
11.9 Hz, 6H), 4.36 (d, J=15.5 Hz, 2H), 4.03 (d, J=3.6 Hz, 2H), 3.97
(d, J=5.9 Hz, 2H), 3.89-3.78 (m, 4H), 3.71-3.68 (m, 2H), 3.64-3.36
(m, 14H), 2.49 (s, 6H), 2.26-2.19 (m, 2H), 2.13-2.06 (m, 2H),
1.90-1.84 (m, 2H), 1.85-1.79 (m, 2H), 1.61-1.55 (m, 4H), 1.04 (d,
J=3.4 Hz, 18H).
[0340] .sup.13C-NMR (101 MHz, MeOD) .delta.: 174.4, 174.3, 172.1,
171.9, 171.8, 171.7, 153.3, 140.6, 131.1, 130.4, 129.0, 72.3, 71.8,
71.2, 71.1, 70.9, 69.9, 69.4, 68.7, 68.4, 60.8, 58.2, 58.1, 58.0,
43.7, 38.9, 37.2, 37.1, 31.1, 31.0, 27.5, 27.0, 15.4. HRMS: found
1191.6137 [M+H.sup.+].
(S)-1-((2R,3R,4S)-3-Fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)ca-
rbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-aminium chloride
(129)
##STR00054##
[0342] Compound 129 was prepared accordingly to PATENT WO
2018/051107 A1. Analytical data matched those previously
reported.
[0343]
N1,N20-Bis((S)-1-((2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthia-
zol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3-
,6,9,12,15,18-hexaoxaicosanediamide (130)
##STR00055##
[0344] Prepared accordingly to general method F, starting from
compound 129 (16.9 mg, 0.0348 mmol) and
3,6,9,12,15,18-hexaoxaicosanedioic acid (6.17 mg, 0.0174 mmol).
Obtained 7.5 mg (35% yield) as white solid.
[0345] .sup.1H-NMR (400 MHz, MeOD) .delta.: 8.89 (s, 2), 7.46 (d,
J=8.7 Hz, 8H), 4.99 (td, J=3.3, 52.9 Hz, 2H), 4.69 (s, 2H), 4.65
(dd, J=2.9, 21.3 Hz, 2H), 4.60-4.34 (m, 6H), 4.08-4.03 (m, 6H),
3.77-3.59 (m, 22H), 2.49 (s, 6H), 1.06 (s, 18H).
[0346] .sup.19F-NMR (376.45 MHz, MeOD): -201.87 ,.sup.13C-NMR (101
MHz, MeOD) .delta.: 170.9, 170.5, 169.2, 169.1, 151.5, 147.7,
138.6, 130.2, 129.0, 127.5, 94.0, 92.1, 70.9, 70.2, 70.1, 70.1,
69.6, 69.5, 64.4, 64.1, 56.1, 50.9, 42.4, 35.3, 25.5, 14.4. HRMS:
found 1215.5214 [M+H.sup.+].
[0347] Abbreviations
[0348] BAIB, bis-acetoxy iodobenzene;
[0349] CID, chemical inducer of dimerization;
[0350] CRL, Cullin RING ligase;
[0351] DC50, half-degrading concentration;
[0352] DCM, dichloromethane;
[0353] DIPEA, N,N-Diisopropyethylamine;
[0354] DMF, dimethylformamide;
[0355] DMSO, dimethylsulfoxide;
[0356] HATU,
1-[Bis(dimethylamino)methylene]-1H-1,2,3Ytriazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate;
[0357] Hdy-HIF-1.alpha., hydroxylated form of HIF-1.alpha.;
[0358] HIF-1.alpha., hypoxia inducible factor alpha;
[0359] Hyp, hydroxyproline;
[0360] HOAT, 1-Hydroxy-7-azabenzotriazole;
[0361] IAPS, inhibitor of apoptosis proteins;
[0362] ITC, isothermal titration calorimetry;
[0363] LHS, left hand side;
[0364] PEG, polyethylene glycol;
[0365] PHD, prolyl hydroxylase domain-containing protein;
[0366] PPI, protein-protein interaction;
[0367] PROTACS, Proteoysis-Targeting Chimeras;
[0368] RHS, right end side;
[0369] SEC, size exclusion chromatography;
[0370] TEMPO, 2,2,6,6-Tetramethylpiperidin-1-yl)oxyl or
(2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl;
[0371] TFA, trifluoroacetic acid;
[0372] VHL , von Hippel-Lindau;
[0373] HRE, hypoxia response element.
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