Note: Descriptions are shown in the official language in which they were submitted.
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SUBSTITUTED SPIRO DERIVATIVES
FIELD OF THE INVENTION
The present invention relates to pharmaceutical agents useful for therapy
and/or prophylaxis
in a mammal, pharmaceutical composition comprising such compounds, and their
use as
menin/MLL protein/protein interaction inhibitors, useful for treating diseases
such as cancer,
myelodysplastic syndrome (MD S) and diabetes.
BACKGROUND OF THE INVENTION
Chromosomal rearrangements affecting the mixed lineage leukemia gene (MLL;
MLL1;
KAJT2A) result in aggressive acute leukemias across all age groups and still
represent mostly
incurable diseases emphasizing the urgent need for novel therapeutic
approaches. Acute
leukemias harboring these chromosomal translocations of MLL represent as
lymphoid, myeloid
or biphenotypic disease and constitute 5 to 10% of acute leukemias in adults
and approximately
70% in infants (Marschalek, Br J Haematol 2011 152(2), 141-54; Tomizawa et
al., Pediatr
Blood Cancer 2007 49(2), 127-32).
MLL is a histone methyltransferase that methylates histone H3 on lysine 4
(H3K4) and
functions in multiprotein complexes. Use of inducible loss-of-function alleles
of Mill
demonstrated that M111 plays an essential role in sustaining hematopoietic
stem cells (HSCs)
and developing B cells although its histone methyltransferase activity is
dispensable for
hematopoiesis (Mishra et al., Cell Rep 2014. 7(4), 1239-47).
Fusion of MLL with more than 60 different partners has been reported to date
and has been
associated with leukemia formation/progression (Meyer et al., Leukemia 2013.
27, 2165-2176).
Interestingly, the SET (Su(var)3-9, enhancer of zeste, and trithorax) domain
of MILL is not
retained in chimeric proteins but is replaced by the fusion partner (Thiel et
al., Bioessays 2012.
34, 771-80). Recruitment of chromatin modifying enzymes like Dot1L and/or the
pTEFb
complex by the fusion partner leads to enhanced transcription and
transcriptional elongation of
MLL target genes including HOXA genes (e.g. HOXA 9) and the HOX cofactor
AIEISl as the
most prominent ones. Aberrant expression of these genes in turn blocks
hematopoietic
differentiation and enhances proliferation.
Menin which is encoded by the Multiple Endocrine Neoplasia type 1 (MENI) gene
is expressed
ubiquitously and is predominantly localized in the nucleus. It has been shown
to interact with
numerous proteins and is, therefore, involved in a variety of cellular
processes. The best
understood function of menin is its role as an oncogenic cofactor of MILL
fusion proteins. Menin
interacts with two motifs within the N-terminal fragment of MILL that is
retained in all fusion
proteins, MBM1 (menin-binding motif 1) and 1V1BM2 (Thiel et al., Bioessays
2012. 34, 771-
80). Menin/MLL interaction leads to the formation of a new interaction surface
for lens
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epithelium-derived growth factor (LEDGF). Although MLL directly binds to
LEDGF, menin
is obligatory for the stable interaction between MILL and LEDGF and the gene
specific
chromatin recruitment of the MLL complex via the PWWP domain of LEDGF
(Cermakova et
al., Cancer Res 2014. 15, 5139-51; Yokoyama & Cleary, Cancer Cell 2008. 8, 36-
46).
Furthermore, numerous genetic studies have shown that menin is strictly
required for oncogenic
transformation by MILL fusion proteins suggesting the menin/MLL interaction as
an attractive
therapeutic target. For example, conditional deletion of Men] prevents
leukomogenesis in bone
marrow progenitor cells ectopically expressing MILL fusions (Chen et al., Proc
Natl Acad Sci
2006. 103, 1018-23). Similarly, genetic disruption of menin/MLL fusion
interaction by loss-of-
function mutations abrogates the oncogenic properties of the MILL fusion
proteins, blocks the
development of leukemia in vivo and releases the differentiation block of MLL-
transformed
leukemic blasts. These studies also showed that menin is required for the
maintenance of HOX
gene expression by MILL fusion proteins (Yokoyama et al., Cell 2005. 123, 207-
18). In addition,
small molecule inhibitors of menin/MLL interaction have been developed
suggesting
druggability of this protein/protein interaction and have also demonstrated
efficacy in
preclinical models of AML (Borkin et al., Cancer Cell 2015. 27, 589-602;
Cierpicki and
Grembecka, Future Med Chem 2014. 6, 447-462). Together with the observation
that menin is
not a requisite cofactor of MLL1 during normal hem atopoiesi s (Li et al.,
Blood 2013. 122,
2039-2046), these data validate the disruption of menin/MLL interaction as a
promising new
therapeutic approach for the treatment of MLL rearranged leukemia and other
cancers with an
active HOXIMEISI gene signature. For example, an internal partial tandem
duplication (PTD)
within the 5'region of the MLL gene represents another major aberration that
is found
predominantly in de novo and secondary AML as well as myeloid dysplasia
syndromes.
Although the molecular mechanism and the biological function of MLL-PTD is not
well
understood, new therapeutic targeting strategies affecting the menin/MLL
interaction might
also prove effective in the treatment of MILL-PTD-related leukemias.
Furthermore, castration-
resistant prostate cancer has been shown to be dependent on the menin/MLL
interaction (Malik
et al., Nat Med 2015. 21, 344-52).
MILL protein is also known as Hi stone-lysine N-methyltransferase 2A (KMT2A)
protein in
the scientific field (UniProt Accession # Q03164).
Several references describe inhibitors targeting the menin-MILL interaction:
W02011029054,
J Med Chem 2016, 59, 892-913 describe the preparation of thienopyrimidine and
benzodiazepine derivatives; W02014164543 describes thienopyrimidine and
thienopyridine
derivatives; Nature Chemical Biology March 2012, 8, 277-284 and Ren, J.; et
al. Bioorg Med
Cheri Lett (2016), 26(18), 4472-4476 describe thienopyrimidine derivatives; J
Med Chem 2014,
57, 1543-1556 describes hydroxy- and aminomethylpiperidine derivatives, Future
Med Chem
2014, 6, 447-462 reviews small molecule and peptidomimetic compounds;
W02016195776
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describes furo[2,3-d]pyrimidine, 9H-purine, [1,3]oxazolo[5,4-d]pyrimidine,
[1,3]oxazolo[4,5-
d]pyrimidine, [1,3 ]thi azolo[5,4 -d]pyrimi dine,
thieno[2,3 -b]pyri dine and thi eno[2,3-
d]pyrimi dine derivatives; W02016197027 describes
5,6,7, 8-tetrahydropyri do[3,4-
d]pyrimi dine, 5,6,7,8-tetrahydropyrido]4,3-d]pyrimidine,
pyrido[2,3-d]pyrimidine and
quinoline derivatives; and W02016040330 describes thienopyrimidine and
thienopyridine
compounds. W02017192543 describes piperidines as Menin inhibitors.
W02017112768,
W02017207387, W02017214367, W02018053267 and W02018024602 describe inhibitors
of the menin-MLL interaction. W02017161002 and W02017161028 describe
inhibitors of
menin-MLL. W02018050686, W02018050684 and W02018109088 describe inhibitors of
the
menin-MLL interaction. W02018226976 describes methods and compositions for
inhibiting
the interaction of menin with MILL proteins. W02019060365 describes
substituted inhibitors
of menin-MLL. Krivtsov et al., Cancer Cell 2019. No.6 Vol.36, 660-673
describes a menin-
MLL inhibitor.
W02020069027 discloses inhibitors of Menin. W02018175746 discloses methods for
treating hematological malignancies and ewing's sarcoma. W02020045334
discloses
azabicyclic derivative used in pharmaceutical compositions. W02019120209
discloses
substituted heterocyclic compounds as menin/lVILL protein/protein interaction
inhibitors
CN111297863 discloses use of menin-mixed lineage leukemia (MLL) inhibitors.
W02021121327 describes substituted straight chain Spiro derivatives and their
use as
menin/MLL protein/protein interaction inhibitors.
DESCRIPTION OF THE INVENTION
The present invention concerns novel compounds of Formula (I),
4
R---,X1/R3
X2
1
n3(X)n4
n1( )n2
R1 a
(I)
2
L.rLj
---
Rib PSI U
and the tautomers and the stereoisomeric forms thereof, wherein
0
<N> xa xb
R1 a represents -C(=0)- xNR aRxb; or N R R
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R" and 10 are each independently selected from the group consisting of
hydrogen;
C3_6cycloalky1; C3_4alkyl, C3_4alkyl substituted with 1, 2 or 3 halo atoms;
and C3_4alkyl
substituted with one -OH, -0C3_4alkyl, or NR11eR11d;
lb
x represents F or Cl;
Y1 represents -CleaR513 - , -0-, - S - , or
R2 is selected from the group consisting of hydrogen, halo, Ci_4alkyl, -0-
Ci_4alky1, and
-NR7aleb;
U1 and U2 each independently represent N or CH;
nl, n2, n3 and n4 are each independently selected from 1 and 2;
X1 represents CH, and X2 represents N;
R4 represents Ci_salkyl,
ss.,.
,.; or ''= ;
R'a, RTh, R', R7a, and RTh, are each independently selected from the group
consisting of
hydrogen, CiAalkyl and C3_6cycloalkyl;
R3 is selected from the group consisting of Het', Het2, Cy2, and -Ci_6alkyl-
NR"Rxd;
R' represents Cy'; Het5; -C1_6a1ky1-Cy1; -C1_6alkyl-Het3; -Ci_6a1ky1-Het4;
or -Ci_6a1ky1-phenyl;
It'd represents hydrogen, C1_4a1ky1, or Ci_4alkyl substituted with one, two or
three sub stituents
selected from the group consisting of halo, -OH, -0-C3_4a1ky1, and cyano,
or We and It'd are taken together to form together with the N-atom to which
they are attached
a 4- to 7-membered monocyclic fully saturated heterocyclyl containing one N-
atom and
optionally one additional heteroatom selected from 0, S, and N, wherein said S-
atom might
be substituted to form S(=0) or S(=0)2; wherein said heterocyclyl is
optionally substituted
with one, two or three substituents selected from the group consisting of
halo, -OH, -O-Ci and cyano,
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2; or a bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl
containing one
N-atom and optionally one or two additional heteroatoms each independently
selected from
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0, S, and N, wherein said S-atom might be substituted to form S(=0) or S(=0)2;
wherein said heterocyclyl is optionally substituted on one nitrogen with a
substituent selected
from the group consisting of R6 and -C(=0)-R8; and wherein said heterocyclyl
is optionally
substituted on one or two carbon atoms with in total one, two, three or four
substituents each
independently selected from the group consisting of halo, R6, Het' Het6b, ,
Ci_4alkyl, oxo, -
NR9aR9b and -OH,
Het2 represents C-linked pyrazolyl or triazolyl; which is substituted on one
nitrogen atom
with R6a;
R6 is selected from the group consisting of
Het3; -C(=0)-NH-R8;
C1_6alkyl optionally substituted with one or two substituents each
independently selected from
the group consisting of Het3, Het4, Het", Het6b, Cy', -CN, -OH,
-0-C1_4alkyl, -C(=0)-NH-C1_4a1kyl, -C(=0)-NH-Ci_4alkyl-C3_6cycloalkyl, -C(=0)-
0H, -
NRI laR1 lb and --I\TH_=-, (_
0)2-Ci_4alkyl; and
C3_6cycloalky1 optionally substituted by one or two substituents each
independently selected
from the group consisting of -CN, -OH, -0-C1_4alkyl, -C(=0)-NH-Ci_4a1kyl,
-NH-S(=0)2-Ci_4alkyl, and C1_4alkyl optionally substituted with one
substituent selected from
the group consisting of OH, -0-C14alkyl, -C(=0)-NH-C1_4alkyl and
-NH-S(=0)2-Ci_4alkyl;
R6a represents C16alkyl substituted with one substituent selected from the
group consisting of
_NR1 laR1 lb, Het3', and Het",
Rs represents Ci_6a1ky1 optionally substituted with one, two or three
substituents each
independently selected from -OH, halo, cyano, -
NRilaR1 lb, He.t3a,
and Het";
Het3 and Het' each independently represent a monocyclic C-linked 4- to 7-
membered fully
saturated heterocyclyl containing one, two or three heteroatoms each
independently selected
from 0, S, and N, wherein said S-atom might be substituted to form S(=0) or
S(=0)2; or a
bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl containing
one, two or three
heteroatoms each independently selected from 0, S, and N, wherein said S-atom
might be
substituted to form S(=0) or S(=0)2;
wherein said heterocyclyl is optionally substituted on one carbon atom with
Ci_4alkyl, halo, -
OH, -
NRilaR1 lb, or oxo; and wherein said heterocyclyl is optionally substituted on
one
nitrogen atom with C1_4alkyl;
Het3a and Hee' each independently represent a monocyclic C-linked 4- to 7-
membered fully
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saturated heterocyclyl containing one N-atom, and optionally one or two
additional
heteroatoms each independently selected from 0, S, and N, wherein said S-atom
might be
substituted to form S(=0) or S(=0)2; or a bicyclic C-linked 6- to 11-membered
fully saturated
heterocyclyl containing one N-atom, and optionally one or two additional
heteroatoms each
independently selected from 0, S, and N, wherein said S-atom might be
substituted to form
S(=0) or S(=0)2;
wherein said heterocyclyl is optionally substituted on one carbon atom with
C1_4alkyl, halo, -
OH, -
NR1 laR1 lb, or oxo; and wherein said heterocyclyl is optionally substituted
on one
nitrogen atom with Ci_4alkyl;
Hee and Het7 each independently represent a monocyclic C-linked 5- or 6-
membered
aromatic ring containing one, two, three or four heteroatoms each
independently selected
from 0, S, and N; wherein said 5-membered aromatic ring is optionally
substituted on one
nitrogen atom with C1_4alkyl; and wherein said 5- or 6-membered aromatic ring
is optionally
substituted on one carbon atom with -OH;
Het' and Het' each independently represent a monocyclic N-linked 4- to 7-
membered fully
saturated heterocyclyl containing one N-atom and optionally one or two
additional
heteroatoms each independently selected from 0, S, and N, wherein said S-atom
might be
substituted to form S(=0) or S(=0)2; wherein said heterocyclyl is optionally
substituted on
one or two carbon atoms with in total one, two, three or four substituents
each independently
selected from the group consisting of halo, -OH, oxo, -(C=0)-
ONR1 aRl0b,0-C3_6cycloalkyl, -
S(=0)2-Ci_4alkyl, cyano, Ch4alkyl, -Ci_4alkyl-OH, -0-C1_4alkyl,
_o_(C=0)_NR10aR101), and
-0-(C=0)-C1_4alkyl; and wherein said heterocyclyl is optionally substituted on
one nitrogen
with a sub stituent selected from the group consisting of -C(=0)-Ci_4alkyl and
-(C=0)-NR'0aRlOb;
Het
each independently represent a monocyclic N-linked 4- to 7-membered fully
saturated
heterocyclyl containing two N-atoms and optionally one additional heteroatom
selected from
0, S, and N, wherein said S-atom might be substituted to form S(=0) or S(=0)2;
wherein said
heterocyclyl is optionally substituted on one or two carbon atoms with in
total one, two, three
or four substituents each independently selected from the group consisting of
halo, -OH, oxo,
-(C=0)-NR'OaRl0b,
-0-C3_6cycloalkyl, -S(=0)2-C1_4a1ky1, cyano, Ci_4alkyl, Ci_4alkyl-OH, -0-
Ci_4alkyl,
-0-(C=0)-
NR 01 aR1013, and -0-(C=0)-Ci_4alkyl; and wherein said heterocyclyl is
optionally
substituted on one nitrogen with a substituent selected from the group
consisting of -C(=0)-
C1_4alkyl and -(C=0)-NRimaRiob;
Het' represents a bicyclic N-linked 6- to 11-membered fully saturated
heterocyclyl
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containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2; wherein said heterocyclyl is optionally substituted on one or two
carbon atoms with
in total one or two sub stituents each independently selected from the group
consisting of Ci_
4a1ky1, -OH, oxo, -(C=0)-N1R10aR1013, _
NH-C(=0)-C1_4alkyl,
-NH-C(=0)-Cy3, and -0-C1_4alkyl; and wherein said heterocyclyl is optionally
substituted on
one nitrogen with a substituent selected from the group consisting of -C(=0)-
C1_4alkyl, -
C(=0)-Cy3, -(C=0)-C1_4alkyl-OH, -C(=0)-Ci_4alkyl-O-Ci_4alkyl,
-C(=0)-Ci_4alkyl-NR'laR11b, and Ci_4alkyl;
Cy' represents C3_6cycloalkyl optionally substituted with one, two or three
substituents
selected from the group consisting of -OH, -NH-C(=0)-C1_4alkyl,
-NH-S(=0)2-Ci_4alkyl, -S(=0)2-C1_4alkyl, and -0-Ci_4alkyl,
Cy 2 represents C3_7cycloalkyl substituted with one or two substituents each
independently
selected from the group consisting of -NleaRTh, Het", Het', and Ci_6alkyl
substituted with
one or two substituents each independently selected from the group consisting
of Het', Het',
Het', and -NR9aR9b; and said C3_7cycloalky1 is optionally substituted with one
or two
additional substituents each independently selected from the group consisting
of halo, R6,
C1_4alkyl, and -OH;
Cy3 represents C3_7cycloalkyl; wherein said C3_7cycloalkyl is optionally
substituted with one,
two or three halo substituents;
R9a and R9b are each independently selected from the group consisting of
hydrogen,
Ci_4alkyl, C3_6cycloalkyl, Hee, -C1_4alkyl-R16, -C(=0)-Ci_4alkyl-Het3a; -C(=O)-
R'4;
C3_6cycloalkyl substituted with one, two or three substituents selected from
the group
consisting of halo, -OH, -0-C1_4alkyl, -NR1laRllb, and cyano, and
Ci_4alkyl substituted with one, two or three substituents selected from the
group consisting of
halo, -OH, -0-Ci_4alkyl, -
NRilaRllb, and cyano;
Rua., R13a, R13b, R15a, R15b, R17a, and Itl7b are each independently
selected from the group
consisting of hydrogen and Ci_4alkyl,
Rile and R' are each independently selected from the group consisting of
hydrogen,
C1_6alkyl, and -C(=0)-Ci_4alkyl,
14
-
tc represents Het'; Hee', or Ci_4alkyl substituted with one, two or
three substituents
selected from the group consisting of 4R13aRl3b and Het';
16
I( represents -C(=0)-NR17aRl7b, _S(=0)2-C1_4alkyl, Het5, Het7, or Het8;
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and the pharmaceutically acceptable salts and the solvates thereof.
The present invention al so relates to a pharmaceutical composition comprising
a therapeutically
effective amount of a compound of Formula (I), a pharmaceutically acceptable
salt, or a solvate
thereof, and a pharmaceutically acceptable carrier or ex ci pi ent.
Additionally, the invention relates to a compound of Formula (T), a
pharmaceutically acceptable
salt, or a solvate thereof, for use as a medicament, and to a compound of
Formula (I), a
pharmaceutically acceptable salt, or a solvate thereof, for use in the
treatment or in the
prevention of cancer, myelodysplastic syndrome (MDS) and diabetes.
In a particular embodiment, the invention relates to a compound of Formula
(I), a
pharmaceutically acceptable salt, or a solvate thereof, for use in the
treatment or in the
prevention of cancer.
In a specific embodiment said cancer is selected from leukemias, myeloma or a
solid tumor
cancer (e.g. prostate cancer, lung cancer, breast cancer, pancreatic cancer,
colon cancer, liver
cancer, melanoma and glioblastoma, etc.). In some embodiments, the leukemias
include acute
leukemias, chronic leukemias, myeloid leukemias, myelogeneous leukemias,
lymphoblastic
leukemias, lymphocytic leukemias, Acute myelogeneous leukemias (AML), Chronic
myelogenous leukemias (CML), Acute lymphoblastic leukemias (ALL), Chronic
lymphocytic
leukemias (CLL), T cell prolymphocytic leukemias (T-PLL), Large granular
lymphocytic
leukemia, Hairy cell leukemia (HCL), MLL-rearranged leukemias, MLL-PTD
leukemias, MILL
amplified leukemias, MILL-positive leukemias, leukemias exhibiting HOXIMEIS1
gene
expression signatures etc.
The invention also relates to the use of a compound of Formula (I), a
pharmaceutically
acceptable salt, or a solvate thereof, in combination with an additional
pharmaceutical agent for
use in the treatment or prevention of cancer, myelodysplastic syndrome (MD S)
and diabetes.
Furthermore, the invention relates to a process for preparing a pharmaceutical
composition
according to the invention, characterized in that a pharmaceutically
acceptable carrier is
intimately mixed with a therapeutically effective amount of a compound of
Formula (I), a
pharmaceutically acceptable salt, or a solvate thereof.
The invention also relates to a product comprising a compound of Formula (I),
a
pharmaceutically acceptable salt, or a solvate thereof, and an additional
pharmaceutical agent,
as a combined preparation for simultaneous, separate or sequential use in the
treatment or
prevention of cancer, myelodysplastic syndrome (MDS) and diabetes.
Additionally, the invention relates to a method of treating or preventing a
cell proliferative
disease in a warm-blooded animal which comprises administering to the said
animal an
effective amount of a compound of Formula (I), a pharmaceutically acceptable
salt, or a solvate
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thereof, as defined herein, or a pharmaceutical composition or combination as
defined herein.
DETAILED DESCRIPTION OF THE INVENTION
The term 'halo' or 'halogen' as used herein represents fluor , chloro, bromo
and iodo.
The prefix 'C,' (where x and y are integers) as used herein refers to the
number of carbon
atoms in a given group. Thus, a C1_6alky1 group contains from 1 to 6 carbon
atoms, and so on.
The term `Ci_4a1kyr as used herein as a group or part of a group represents a
straight or
branched chain saturated hydrocarbon radical having from 1 to 4 carbon atoms,
such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.
Similar, the term `Ci_6alkyr as used herein as a group or part of a group
represents a straight or
branched chain saturated hydrocarbon radical having from 1 to 6 carbon atoms,
such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl and
the like.
The term C3_6cycloalkyl' as used herein as a group or part of a group defines
a saturated,
cyclic hydrocarbon radical having from 3 to 6 carbon atoms, such as
cyclopropyl, cyclobutyl,
cyclopentyl and cyclohexyl.
The term C3_7cycloalkyl' as used herein as a group or part of a group defines
a saturated,
cyclic hydrocarbon radical having from 3 to 7 carbon atoms, such as
cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl and cycloheptyl.
It will be clear for the skilled person that S(=0)2 or SO2 represents a
sulfonyl moiety.
It will be clear for the skilled person that CO or C(=0) represents a carbonyl
moiety.
It will be clear for the skilled person that a group such as -CRR- represents
R R
-C-
. An example of such a group is -CR-SaR-5b-.
It will be clear for the skilled person that a group such as -NR- represents -
N-
. An example
of such a group is -NR-.
The term `monocyclic C-linked 4- to 7-membered fully saturated heterocyclyl
containing one
N-atom, and optionally one or two additional heteroatoms each independently
selected from 0,
S, and N', defines a fully saturated, cyclic hydrocarbon radical having from 4
to 7 ring members
and containing 1 nitrogen atom and optionally one or two additional
heteroatoms each
independently selected from 0, S, and N, such as for example C-linked
azetidinyl, C-linked
pyrrolidinyl, C-linked morpholinyl and C-linked piperidinyl. The term
`monocyclic N-linked
4- to 7-membered fully saturated heterocyclyl containing one N-atom and
optionally one or two
additional heteroatoms each independently selected from 0, S, and N', is
defined similar but is
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attached to the remainder of the molecule of formula (I) via a nitrogen atom
Examples are N-
linked azetidinyl, N-linked pyrrolidinyl, N-linked morpholinyl, N-linked
thiomorpholinyl, N-
linked piperazinyl, N-linked 1,4-diazepanyl, and N-linked piperidinyl. Two R
groups taken
together to form together with the N-atom to which they are attached a 4- to 7-
membered
monocyclic fully saturated heterocyclyl containing one N-atom and optionally
one additional
heteroatom selected from 0, S, and N, are defined similar.
The term `monocyclic C-linked 4- to 7-membered fully saturated heterocyclyl
containing one,
two or three heteroatoms each independently selected from 0, S, and N',
defines a fully
saturated, cyclic hydrocarbon radical having from 4 to 7 ring members and
containing one, two
or three heteroatoms each independently selected from 0, S, and N, such as for
example C-
linked azetidinyl, C-linked pyrrolidinyl, C-linked morpholinyl, C-linked
tetrahydrofuranyl, C-
linked thiolanyl, C-linked oxetanyl, C-linked thietanyl, C-linked
tetrahydropyranyl, and C-
linked piperidinyl. The term cmonocyclic N-linked 4- to 7-membered fully
saturated
heterocyclyl containing two N-atoms and optionally one additional heteroatom
selected from
0, S, and N', defines a fully saturated, cyclic hydrocarbon radical having
from 4 to 7 ring
members and containing 2 nitrogen atoms and optionally one additional
heteroatom selected
from 0, S, and N, such as for example N-linked piperazinyl, and N-linked 1,4-
diazepanyl.
For clarity, the 4- to 7-membered fully or partially saturated heterocyclyls
have from 4 to 7
ring members including the heteroatoms.
Non-limiting examples of `monocyclic 5- or 6-membered aromatic rings
containing one or
two nitrogen atoms and optionally a carbonyl moiety', include, but are not
limited to
pyrazolyl, imidazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1H-
1,2,4-triazolyl, 4H-
1,2,4-triazolyl, 1,2,4-triazinyl, 1,2-dihydro-2-oxo-5-pyrimidinyl, 1,2-dihydro-
2-oxo-6-
pyridinyl, 1,2-dihydro-2-oxo-4-pyridinyl, and 1,6-dihydro-6-oxo-3-pyridazinyl.
Non-limiting examples of `monocyclic C-linked 5-or 6-membered aromatic rings
containing
one, two or three heteroatoms each independently selected from 0, S. and N',
include, but are
not limited to C-linked pyrazolyl, C-linked imidazolyl, C-linked pyridinyl, C-
linked triazolyl,
C-linked pyridazinyl, C-linked pyrimidinyl, C-linked oxazolyl, C-linked
furanyl, C-linked
isothiazolyl, or C-linked pyrazinyl.
Within the context of this invention, bicyclic C-linked 6- to 11-membered
fully saturated
heterocyclyl groups, include fused, spiro and bridged bicycles
Within the context of this invention, bicyclic N-linked 6-to 11-membered fully
saturated
heterocyclyl groups, include fused, Spiro and bridged bicycles.
Fused bicyclic groups are two cycles that share two atoms and the bond between
these atoms.
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Spiro bicyclic groups are two cycles that are joined at a single atom
Bridged bicyclic groups are two cycles that share more than two atoms.
Examples of bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl
containing one
N-atom and optionally one or two additional heteroatoms each independently
selected from
0, S, and N, include, but are not limited to
NH
,
N C H H
NH 1N
0
Of)
and the like.
Examples of bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl
containing one,
two or three heteroatoms each independently selected from 0, S, and N,
include, but are not
limited to
N H H H
N O H H
N H N
OK>
0
J
and the like.
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Examples of bicyclic N-linked 6- to 11-membered fully saturated heterocyclyl
containing one
N-atom and optionally one or two additional heteroatoms each independently
selected from
0, S, and N, include, but are not limited to
0 NO N H
N/ ________________________________________________________________ X:D>
N(r>
NH N )00
----N
H ,7N1
- - -N
H
0
- -N
0
- -
0
and the like
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Whenever substituents are represented by chemical structure, such as for
example
H
represents the bond of attachment to the remainder of the molecule of Formula
(T)
When any variable occurs more than one time in any constituent, each
definition is
independent.
When any variable occurs more than one time in any formula (e.g. Formula (I)),
each
definition is independent.
In this context, it will also be clear that a term like "optionally
substituted with one, two or
three sub stituents selected from the group consisting of' is equivalent to
"optionally
substituted with one, two or three substituents each independently selected
from the group
consisting of'.
In general, whenever the term 'substituted' is used in the present invention,
it is meant, unless
otherwise indicated or clear from the context, to indicate that one or more
hydrogens, in
particular from 1 to 4 hydrogens, more in particular from 1 to 3 hydrogens,
preferably 1 or 2
hydrogens, more preferably 1 hydrogen, on the atom or radical indicated in the
expression
using 'substituted' are replaced with a selection from the indicated group,
provided that the
normal valency is not exceeded, and that the substitution results in a
chemically stable
compound, i.e. a compound that is sufficiently robust to survive isolation to
a useful degree of
purity from a reaction mixture. In a particular embodiment, when the number of
substituents
is not explicitly specified, the number of sub stituents is one.
Combinations of sub stituents and/or variables are permissible only if such
combinations result
in chemically stable compounds. 'Stable compound' is meant to indicate a
compound that is
sufficiently robust to survive isolation to a useful degree of purity from a
reaction mixture.
The skilled person will understand that the term 'optionally substituted'
means that the atom
or radical indicated in the expression using 'optionally substituted' may or
may not be
substituted (this means substituted or unsubstituted respectively).
When two or more substituents are present on a moiety they may, where possible
and unless
otherwise indicated or clear from the context, replace hydrogens on the same
atom or they
may replace hydrogen atoms on different atoms in the moiety.
Within the context of this invention 'saturated' means 'fully saturated', if
not otherwise
specified.
Unless otherwise specified or clear from the context, aromatic rings and
heterocyclyl goups,
can be attached to the remainder of the molecule of Formula (I) through any
available ring
carbon atom (C-linked) or nitrogen atom (N-linked).
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Unless otherwise specified or clear from the context, aromatic rings and
heterocyclyl goups,
may optionally be substituted, where possible, on carbon and/or nitrogen atoms
according to
the embodiments.
The term "subject" as used herein, refers to an animal, preferably a mammal
(e.g. cat, dog,
primate or human), more preferably a human, who is or has been the object of
treatment,
observation or experiment.
The term "therapeutically effective amount" as used herein, means that amount
of active
compound or pharmaceutical agent that elicits the biological or medicinal
response in a tissue
system, animal or human that is being sought by a researcher, veterinarian,
medicinal doctor
or other clinician, which includes alleviation or reversal of the symptoms of
the disease or
disorder being treated.
The term "composition" is intended to encompass a product comprising the
specified
ingredients in the specified amounts, as well as any product which results,
directly or
indirectly, from combinations of the specified ingredients in the specified
amounts.
The term "treatment", as used herein, is intended to refer to all processes
wherein there may
be a slowing, interrupting, arresting or stopping of the progression of a
disease, but does not
necessarily indicate a total elimination of all symptoms.
The term "compound(s) of the (present) invention" or "compound(s) according to
the
(present) invention" as used herein, is meant to include the compounds of
Formula (I) and the
pharmaceutically acceptable salts, and the solvates thereof.
As used herein, any chemical formula with bonds shown only as solid lines and
not as solid
wedged or hashed wedged bonds, or otherwise indicated as having a particular
configuration
(e.g. R, S) around one or more atoms, contemplates each possible stereoisomer,
or mixture of
two or more stereoisomers.
Hereinbefore and hereinafter, the term "compound(s) of Formula (I)" is meant
to include the
tautomers thereof and the stereoisomeric forms thereof.
The terms "stereoisomers", "stereoisomeric forms" or "stereochemically
isomeric forms"
hereinbefore or hereinafter are used interchangeably.
The invention includes all stereoisomers of the compounds of the invention
either as a pure
stereoisomer or as a mixture of two or more stereoisomers.
Enantiomers are stereoisomers that are non-superimposable mirror images of
each other. A
1:1 mixture of a pair of enantiomers is a racemate or racemic mixture.
Atropisomers (or atropoisomers) are stereoisomers which have a particular
spatial
configuration, resulting from a restricted rotation about a single bond, due
to large steric
hindrance. All atropisomeric forms of the compounds of Formula (I) are
intended to be
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included within the scope of the present invention.
Diastereomers (or diastereoisomers) are stereoisomers that are not
enantiomers, i.e. they are
not related as mirror images. If a compound contains a double bond, the
substituents may be
in the E or the Z configuration.
Sub stituents on bivalent cyclic saturated or partially saturated radicals may
have either the
cis- or trans-configuration; for example if a compound contains a di
substituted cycloalkyl
group, the substituents may be in the cis or trans configuration.
Therefore, the invention includes enantiomers, atropisomers, diastereomers,
racemates, E
isomers, Z isomers, cis isomers, trans isomers and mixtures thereof, whenever
chemically
possible.
The meaning of all those terms, i.e. enantiomers, atropisomers, di
astereomers, racemates, E
isomers, Z isomers, cis isomers, trans isomers and mixtures thereof are known
to the skilled
person.
The absolute configuration is specified according to the Cahn-Ingold-Prelog
system. The
configuration at an asymmetric atom is specified by either R or S. Resolved
stereoisomers
whose absolute configuration is not known can be designated by (+) or
(-) depending on the direction in which they rotate plane polarized light. For
instance,
resolved enantiomers whose absolute configuration is not known can be
designated by (+) or
(-) depending on the direction in which they rotate plane polarized light.
When a specific stereoisomer is identified, this means that said stereoisomer
is substantially
free, i.e. associated with less than 50%, preferably less than 20%, more
preferably less than
10%, even more preferably less than 5%, in particular less than 2% and most
preferably less
than 1%, of the other stereoisomers. Thus, when a compound of Formula (1) is
for instance
specified as (R), this means that the compound is substantially free of the
(S) isomer; when a
compound of Formula (I) is for instance specified as E, this means that the
compound is
substantially free of the Z isomer; when a compound of Formula (I) is for
instance specified
as cis, this means that the compound is substantially free of the trans
isomer.
Some of the compounds according to Formula (I) may also exist in their
tautomeric form.
Such forms in so far as they may exist, although not explicitly indicated in
the above Formula
(I) are intended to be included within the scope of the present invention.
For example
N-NH N-N ______ OH
___________________________ 0
is equivalent to
It follows that a single compound may exist in both stereoisomeric and
tautomeric form.
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Pharmaceutically acceptable salts include acid addition salts and base
addition salts. Such
salts may be formed by conventional means, for example by reaction of a free
acid or a free
base form with one or more equivalents of an appropriate base or acid,
optionally in a solvent,
or in a medium in which the salt is insoluble, followed by removal of said
solvent, or said
medium, using standard techniques (e.g. in vacuo, by freeze-drying or by
filtration). Salts may
also be prepared by exchanging a counter-ion of a compound of the invention in
the form of a
salt with another counter-ion, for example using a suitable ion exchange
resin.
The pharmaceutically acceptable salts as mentioned hereinabove or hereinafter
are meant to
comprise the therapeutically active non-toxic acid and base salt forms which
the compounds
of Formula (I) and solvates thereof, are able to form.
Appropriate acids comprise, for example, inorganic acids such as hydrohalic
acids, e.g.
hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like
acids; or organic
acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic,
oxalic (i.e.
ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric,
malic, tartaric, citric,
methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic,
salicylic, p-
aminosalicylic, pamoic and the like acids. Conversely said salt forms can be
converted by
treatment with an appropriate base into the free base form.
The compounds of Formula (I) and solvates thereof containing an acidic proton
may also be
converted into their non-toxic metal or amine salt forms by treatment with
appropriate organic
and inorganic bases.
Appropriate base salt forms comprise, for example, the ammonium salts, the
alkali and earth
alkaline metal salts, e.g. the lithium, sodium, potassium, cesium, magnesium,
calcium salts
and the like, salts with organic bases, e.g. primary, secondary and tertiary
aliphatic and
aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine,
the four
butylamine isomers, dimethylamine, diethylamine, diethanolamine,
dipropylamine,
diisopropylamine, di-n-butylamine, pyrroli dine, piperidine, morpholine,
trimethylamine,
triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and
isoquinoline; the
benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino
acids such as,
for example, arginine, lysine and the like. Conversely the salt form can be
converted by
treatment with acid into the free acid form.
The term solvate comprises the solvent addition forms as well as the salts
thereof, which the
compounds of Formula (I) are able to form. Examples of such solvent addition
forms are e.g.
hydrates, alcoholates and the like.
The compounds of the invention as prepared in the processes described below
may be
synthesized in the form of mixtures of enantiomers, in particular racemic
mixtures of
enantiomers, that can be separated from one another following art-known
resolution
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procedures. A manner of separating the enantiomeric forms of the compounds of
Formula (I),
and pharmaceutically acceptable salts, and solvates thereof, involves liquid
chromatography
using a chiral stationary phase. 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 stereospecifically.
Preferably if a specific
stereoisomer is desired, said compound would be synthesized by stereospecific
methods of
preparation. These methods will advantageously employ enantiomerically pure
starting
materials.
The term "enantiomerically pure" as used herein means that the product
contains at least 80%
by weight of one enantiomer and 20% by weight or less of the other enantiomer.
Preferably the
product contains at least 90% by weight of one enantiomer and 10% by weight or
less of the
other enantiomer. In the most preferred embodiment the term "enantiomerically
pure" means
that the composition contains at least 99% by weight of one enantiomer and 1%
or less of the
other enantiomer.
The present invention also embraces isotopically-labeled compounds of the
present invention
which are identical to those recited herein, but for the fact that one or more
atoms are replaced
by an atom having an atomic mass or mass number different from the atomic mass
or mass
number usually found in nature (or the most abundant one found in nature).
All isotopes and isotopic mixtures of any particular atom or element as
specified herein are
contemplated within the scope of the compounds of the invention, either
naturally occurring
or synthetically produced, either with natural abundance or in an isotopically
enriched form.
Exemplary isotopes that can be incorporated into compounds of the invention
include isotopes
of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine
and iodine, such
as 2H, 3H, 11c, 13c, 14c , 13N, 150, 170, 180, 32Fb, 3313, 35s, 18F, 36c1,
1221, 1231, 1251, 131=,
1 75Br,
76Br, .77Br and 'Br. Preferably, the isotope is selected from the group of 2H,
3H, "C and "F.
More preferably, the isotope is 2H. In particular, deuterated compounds are
intended to be
included within the scope of the present invention.
Certain isotopically-labeled compounds of the present invention (e.g., those
labeled with 3H
and "C) may be useful for example in substrate tissue distribution assays.
Tritiated (3H) and
carbon-14 ("C) isotopes are useful for their ease of preparation and
detectability. Further,
substitution with heavier isotopes such as deuterium (i.e., 2H) may afford
certain therapeutic
advantages resulting from greater metabolic stability (e.g., increased in vivo
half-life or
reduced dosage requirements) and hence may be preferred in some circumstances.
Positron
emitting isotopes such as 150, 13N, "C and 18F are useful for positron
emission tomography
(PET) studies. PET imaging in cancer finds utility in helping locate and
identify tumours,
stage the disease and determine suitable treatment. Human cancer cells
overexpress many
receptors or proteins that are potential disease-specific molecular targets.
Radiolabelled
tracers that bind with high affinity and specificity to such receptors or
proteins on tumour
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cells have great potential for diagnostic imaging and targeted radionuclide
therapy (Charron,
Carlie L. et al. Tetrahedron Lett. 2016, 57(37), 4119-4127). Additionally,
target-specific PET
radiotracers may be used as biomarkers to examine and evaluate pathology, by
for example,
measuring target expression and treatment response (Austin R. et al. Cancer
Letters (2016),
doi: 10.1016/j.canlet.2016.05.008).
The present invention relates in particular to compounds of Formula (I) as
defined herein, and
the tautomers and the stereoisomeric forms thereof, wherein
0
RxaRxb
R1 represents -C(=0)-NRxa
Rxb; or
R" and Wb are each independently selected from the group consisting of
hydrogen;
C3_6cycloalkyl; Ci_4alkyl; and Ci-ialkyl substituted with 1, 2 or 3 halo
atoms;
Rib represents F or Cl;
Y1 represents -CR5aR5b-, -0-, -S-, or -NR5'-;
R2 is selected from the group consisting of hydrogen, halo, Ci-ialkyl, -0-
Ci_4alkyl, and
-NR7aRM ;
and U2 each independently represent N or CH;
nl, n2, n3 and n4 are each independently selected from 1 and 2;
X1 represents CH, and X2 represents N;
R4 represents Ci_salkyl;
(1:).
s.,.
or
R5a, R5b, R5e, R7a, and km, are each independently selected from the group
consisting of
hydrogen, Ci_4alkyl and C3_6cycloalkyl;
R3 is selected from the group consisting of Het', Het2, Cy2, and -
C1_6alkyl_NRxeRxd;
R' represents Cy'; Het5; -Ci_ 6alkyl-Cyl; -CI -6alkyl-Het3; -Ci_6a1ky1-Het4;
or -C1_6alkyl-phenyl;
It'd represents hydrogen; Ci_4a1ky1; or Ci_4alkyl substituted with one, two or
three sub stituents
selected from the group consisting of halo, -OH, -0-Ci_4a1ky1, and cyano;
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or R" and IV' are taken together to form together with the N-atom to which
they are attached
a 4- to 7-membered monocyclic fully saturated heterocyclyl containing one N-
atom and
optionally one additional heteroatom selected from 0, S, and N, wherein said S-
atom might
be substituted to form S(=0) or S(=0)2; wherein said heterocyclyl is
optionally substituted
with one, two or three substituents selected from the group consisting of
halo, -OH,
-0-Ci_4alkyl, and cyano,
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2, or a bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl
containing one
N-atom and optionally one or two additional heteroatoms each independently
selected from
0, S, and N, wherein said S-atom might be substituted to form S(=0) or S(=0)2;
wherein said heterocyclyl is optionally substituted on one nitrogen with a
substituent selected
from the group consisting of R6 and -C(=0)-1e; and wherein said heterocyclyl
is optionally
substituted on one or two carbon atoms with in total one, two, three or four
substituents each
independently selected from the group consisting of halo, R6, Het6a, Het6b,
C1_4alkyl, oxo, -
NR9aR" and -OH,
Het2 represents C-linked pyrazolyl or triazolyl; which is substituted on one
nitrogen atom
with lea;
R6 is selected from the group consisting of
Het3; -C(=0)-NH-R8;
C1_6alkyl optionally substituted with one or two substituents each
independently selected from
the group consisting of Het3, Hee, Het6a, Het6b, Cy', -CN, -OH, -0-C1_4alkyl, -
C(=0)-NH-C1-
4a1ky1, -C(=0)-NH-Ci_4alkyl-C3_6cycloalkyl, -C(=0)-0H, -
NR 11 aR1 lb and 4,414_,-,
012-C1-
4alkyl, and
C3_6cycloalkyl optionally substituted by one or two substituents each
independently selected
from the group consisting of -CN, -OH, -0-C1_4alkyl, -C(=0)-NH-Ci_4a1kyl,
-NH-S(=0)2-Ci_4alkyl, and Ci_4alkyl optionally substituted with one
substituent selected from
the group consisting of OH, -0-C1_4alkyl, -C(=0)-NH-C1_4alkyl and
-NH-S(=0)2-Ci_4alkyl,
-rs 6a
K represents C1-6alkyl substituted with one substituent selected from the
group consisting of
_NR1 laR1 lb, He, 3a,
t and Het6a,
R8 represents C1_6a1kyl substituted with one substituent selected from the
group consisting of -
OH, -
NR1 laR1 lb, Het3', and Het6a;
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Het3 and Het5 each independently represent a monocyclic C-linked 4- to 7-
membered fully
saturated heterocyclyl containing one, two or three heteroatoms each
independently selected
from 0, S, and N, wherein said S-atom might be substituted to form S(=0) or
S(=0)2; or a
bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl containing
one, two or three
heteroatoms each independently selected from 0, S, and N, wherein said S-atom
might be
substituted to form S(=0) or S(=0)2;
wherein said heterocyclyl is optionally substituted on one carbon atom with
C1_4alkyl, halo, -
OH, -
NR1 laR1 lb, or oxo; and wherein said heterocyclyl is optionally substituted
on one
nitrogen atom with Ci_4alkyl;
Hee' and Hee' each independently represent a monocyclic C-linked 4- to 7-
membered fully
saturated heterocyclyl containing one N-atom, and optionally one or two
additional
heteroatoms each independently selected from 0, S, and N, wherein said S-atom
might be
substituted to form S(=0) or S(=0)2; or a bicyclic C-linked 6- to 11-membered
fully saturated
heterocyclyl containing one N-atom, and optionally one or two additional
heteroatoms each
independently selected from 0, S, and N, wherein said S-atom might be
substituted to form
S(=0) or S(=0)2;
wherein said heterocyclyl is optionally substituted on one carbon atom with CI-
4alkyl, halo, -
OH, _NRI aR1 lb, or oxo; and wherein said heterocyclyl is optionally
substituted on one
nitrogen atom with C1_4alkyl;
Het4 and Het7 each independently represent a monocyclic C-linked 5- or 6-
membered
aromatic ring containing one, two or three heteroatoms each independently
selected from 0,
S, and N; wherein said 5-membered aromatic ring is optionally substituted on
one nitrogen
atom with C1_4alkyl; and wherein said 5- or 6-membered aromatic ring is
optionally
substituted on one carbon atom with -OH;
Het6a and Het' each independently represent a monocyclic N-linked 4- to 7-
membered fully
saturated heterocyclyl containing one N-atom and optionally one or two
additional
heteroatoms each independently selected from 0, S, and N, wherein said S-atom
might be
substituted to form S(=0) or S(=0)2; wherein said heterocyclyl is optionally
substituted on
one or two carbon atoms with in total one, two, three or four substituents
each independently
selected from the group consisting of halo, -OH, oxo, -(C=0)-
ONR1 aRl0b,0-C3_6cycloalkyl, -
S(=0)2-C1_4a1ky1, cyano, Ci_4alkyl, -Ci_4alkyl-OH, -0-C1_4alkyl, -0-(C=0)-
NR10aRlOb, and
-0-(C=0)-Ci_4alkyl; and wherein said heterocyclyl is optionally substituted on
one nitrogen
with a substituent selected from the group consisting of -C(=0)-Ci_4alkyl and
-(C=0)-NR10aRlOb;
Het
each independently represent a monocyclic N-linked 4- to 7-membered fully
saturated
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heterocyclyl containing two N-atoms and optionally one additional heteroatom
selected from
0, S, and N, wherein said S-atom might be substituted to form S(=0) or S(=0)2;
wherein said
heterocyclyl is optionally substituted on one or two carbon atoms with in
total one, two, three
or four substituents each independently selected from the group consisting of
halo, -OH, oxo,
-(C=0)-NR1 OaR101),
-0-C3_6cycloalkyl, -S(=0)2-C1_4a1ky1, cyano, Ci_4alkyl, Ci_4alkyl-OH, -0-C
14a1ky1,
-0-(C=0)-
NR10aR1 013, and -0-(C=0)-C1_4alkyl; and wherein said heterocyclyl is
optionally
substituted on one nitrogen with a substituent selected from the group
consisting of -C(=0)-
C1_4alkyl and -(C=0)-NR10aRiob;
Het" represents a bicyclic N-linked 6- to 11-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2; wherein said heterocyclyl is optionally substituted on one carbon atom
with a
substituent selected from the group consisting of -OH, oxo,
-(C=0)-NR'OaRlOb, -NH-C(=0)-Ci_4a1ky1, -NH-C(=0)-Cy3, and -0-C1_4a1ky1, and
wherein
said heterocyclyl is optionally substituted on one nitrogen with a substituent
selected from the
group consisting of -C(=0)-Ci_4alkyl, -C(=0)-Cy3, and C1_4a1ky1;
Cy' represents C3_6cycloalkyl optionally substituted with one, two or three
substituents
selected from the group consisting of -OH, -NH-C(=0)-C1_4alkyl, C1_4alkyl,
-NH-S(=0)2-C1_4alkyl, -S(=0)2-C1_4alkyl, and -0-C1_4alkyl,
Cy2 represents C3_7cycloalkyl substituted with one or two substituents each
independently
selected from the group consisting of -NleaR", Het", Het', and Ci_6alkyl
substituted with
one or two substituents each independently selected from the group consisting
of Het3', Het6',
Het', and -NleaR", and said C3_7cycloalkyl is optionally substituted with one
or two
additional substituents each independently selected from the group consisting
of halo, R6,
C1_4alkyl, and -OH;
Cy3 represents C3_7cycloalkyl; wherein said C3_7cycloa1kyl is optionally
substituted with one,
two or three halo substituents,
R9a and R" are each independently selected from the group consisting of
hydrogen;
C1_4alkyl; C3_6cycloa1kyl, Het5; -C1_4alkyl-R16; -C(=0)-Ci_4alkyl-Het3a; -
C(=0)-R1-4;
C3_6cycloa1kyl substituted with one, two or three substituents selected from
the group
consisting of halo, -OH, -0-C1_4alkyl, and cyano, and
C1_4alkyl substituted with one, two or three substituents selected from the
group consisting of
halo, -OH, -0-Ci_4alkyl, and cyano;
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Rioa, Riob, Riia, let), R13a, Ri3b, Risa, Ri5b, R17a, and Rim are each
independently selected
from the group consisting of hydrogen and Ci-lalkyl;
14
tc represents Het''; Het; or Ct-talkyl substituted with one, two or
three substituents
selected from the group consisting of _NR13aR1313 and Het8a;
1-c - 16
represents -C(=0)-
NR17aRl7b, _S(=0)2-C1_4a11cy1, Het', Het', or Het';
and the pharmaceutically acceptable salts and the solvates thereof.
The present invention relates in particular to compounds of Formula (I) as
defined herein, and
the tautomers and the stereoisomeric forms thereof, wherein
Rla represents -C(=0)-
NRxaRxb;
R' and Wb are each independently selected from the group consisting of
C3_6cycloalkyl; C1_4a1ky1; and CI-talky] substituted with 1, 2 or 3 halo
atoms;
R"
represents F;
Y1 represents -0-;
R2 represents hydrogen;
U1 and U2 each independently represent N or CH;
nl, n2, n3 and n4 are each independently selected from 1 and 2;
X1 represents CH, and X2 represents N;
R4 represents Ci_salkyl;
Nor ''=
R3 is selected from the group consisting of Het' and Cy2;
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is optionally substituted on one nitrogen with a
substituent selected
from the group consisting of R6 and -C(=0)-W; and wherein said heterocyclyl is
optionally
substituted on one or two carbon atoms with in total one, two, three or four
halo substituents;
R6 is selected from the group consisting of Het3;
C1_6alkyl optionally substituted with one or two substituents each
independently selected from
the group consisting of Het3, Heft, Het6a, Cy', -OH, -0-Ct-4a1ky1, -C(=0)-NH-
C1_4alky1, -
C(=0)-NH-C1_4alkyl-C3_6cyc1oalkyl, and -NH-S(=0)2-C1_4alkyl;
R8 represents C1_6a1kyl substituted with one substituent selected from the
group consisting of -
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OH and -NR11111 lb,
Het3 and Het5 each independently represent a monocyclic C-linked 4- to 7-
membered fully
saturated heterocyclyl containing one, two or three heteroatoms each
independently selected
from 0, S, and N, wherein said S-atom might be substituted to form S(=0) or
S(=0)2;
wherein said heterocyclyl is optionally substituted on one carbon atom with -
OH or oxo;
Het4 represents a monocyclic C-linked 5- or 6-membered aromatic ring
containing one, two or
three heteroatoms each independently selected from 0, S, and N; wherein said 5-
or 6-
membered aromatic ring is optionally substituted on one carbon atom with -OH;
Het' represents a monocyclic N-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2; wherein said heterocyclyl is optionally substituted on one or two
carbon atoms with
in total one, two, three or four substituents each independently selected from
the group
consisting of oxo, -S(=0)2-C1_4alkyl, and -0-Ci_4alkyl; and wherein said
heterocyclyl is
optionally substituted on one nitrogen with
-C(=0)-Ci_4a1ky1;
Heel' represents a bicyclic N-linked 6- to 11-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2, wherein said heterocyclyl is optionally substituted on one carbon atom
with -(C=0)-
NRioartiob, and wherein said heterocyclyl is optionally substituted on one
nitrogen with -
C(=0)-C1_4a1ky1,
Cy' represents C3_6cycloalkyl optionally substituted with one, two or three
substituents
selected from the group consisting of -OH, -NH-C(=0)-Ci_4alkyl, C1_4alkyl,
-NH-S(=0)2-C1_4a1ky1, and -0-C 1_4a1ky1;
Cy2 represents C3_7cycloalkyl substituted with one or two substituents each
independently
selected from the group consisting of -NleaR9b, Het"; and Heeb;
R9a and R91' are each independently selected from the group consisting of
hydrogen;
Ci_4alkyl; C3_6cycloalkyl, Het5; -Ci_4alkyl-R16; and
C1_4alkyl substituted with one, two or three -0-C1_4alkyl substituents;
R10', R1011, R1 1 a, and R1 lb represent Ci_4alkyl;
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-=-= 16
tc represents Hee;
and the pharmaceutically acceptable salts and the solvates thereof.
The present invention relates in particular to compounds of Formula (I) as
defined herein, and
the tautomers and the stereoisomeric forms thereof, wherein
Ria represents -C(=0)- aNRx Rxb;
It' and Rxb represent C1_4alkyl;
tc represents F;
Y1 represents -0-;
R2 represents hydrogen;
U1 and U2 each independently represent N or CH;
nl, n2, n3 and n4 are each independently selected from 1 and 2;
X' represents CH, and X2 represents N;
R4 represents isopropyl;
K3 is selected from the group consisting of Het' and Cy2;
Het' represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is optionally substituted on one nitrogen with a
substituent selected
from the group consisting of R6;
R6 represents C1_6a1kyl substituted with one Het3;
Het3 represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one, two or three heteroatoms each independently selected from 0,
S, and N,
wherein said S-atom might be substituted to form S(=0) or S(=0)2;
Het6a represents a monocyclic N-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom; wherein said heterocyclyl is optionally substituted on
one carbon
atom with one -0-Ci_4alkyl;
Heeb represents a bicyclic N-linked 6- to 11-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0 and N; wherein said heterocyclyl is optionally substituted on
one carbon
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atom with -(C=0)-
ONR1 aRlOb; and wherein said heterocyclyl is optionally substituted on one
nitrogen with -C(=0)-Ci4alkyl;
Cy2 represents C3_7cycloalkyl substituted with one substituents selected from
the group
consisting of Het' and Het6b;
Rma and Ri" represent C1_4alkyl;
and the pharmaceutically acceptable salts and the solvates thereof.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
Rla represents -C(=0)- aNRx Rxb.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
T's
tc represents F.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R2 represents hydrogen.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein n1 is 1, n2 is 2, n3 is 1,
and n4 is 1.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein Ul represents N
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein Ul represents N, and U2
represents N.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein U' represents CH, and U2
represents N
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
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mentioned in any of the other embodiments, wherein
Y1 represents -0-.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
Y1 represents -0-; and
U2 represents N
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
Y1 represents -0-; and
U1 represents N.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
Y1 represents -0-;
U2 represents N;
tc represents F; and
R2 represents hydrogen.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
Y1 represents -0-,
U1 represents N,
Rib represents F, and
R2 represents hydrogen.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
Y1 represents -0-,
U1 represents N;
lb
Rrepresents F;
R2 represents hydrogen; and
R4 represents isopropyl.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
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mentioned in any of the other embodiments, wherein
R`i represents isopropyl.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R3 represents Het' or Cy2.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R3 represents Cy2
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R3 represents Het'.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2; or a bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl
containing one
N-atom and optionally one or two additional heteroatoms each independently
selected from
0, S, and N, wherein said S-atom might be substituted to form S(-0) or S(=0)2;
wherein said heterocyclyl is optionally substituted on one nitrogen with R6,
and wherein said
heterocyclyl is optionally substituted on one or two carbon atoms with in
total one, two, three
or four substituents each independently selected from the group consisting of
halo, R6, Hee',
Ci_4alkyl, oxo, -NR9aR9b and -OH.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R3 represents He-0;
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom and optionally one or two additional heteroatoms each
independently
selected from 0, S, and N, wherein said S-atom might be substituted to form
S(=0) or
S(=0)2, or a bicyclic C-linked 6- to 11-membered fully saturated heterocyclyl
containing one
N-atom and optionally one or two additional heteroatoms each independently
selected from
0, S, and N, wherein said S-atom might be substituted to form S(=0) or S(=0)2;
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wherein said heterocyclyl is optionally substituted on one nitrogen with R6;
and wherein said
heterocyclyl is optionally substituted on one or two carbon atoms with in
total one, two, three
or four substituents each independently selected from the group consisting of
halo, R6, Het6a,
Het6b, Ci_4alkyl, oxo, -NR9aR9b and -OH.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R6 is selected from the group consisting of
Hct3; -C(=0)-NH-R8;
C1_6alkyl optionally substituted with one or two sub stituents each
independently selected from
the group consisting of Het3, Hee, Het6a, Het6b, Cy', -CN, -OH, -0-C1_4alkyl, -
C(=0)-NH-Ci-
4alkyl, -C(=0)-NII-Ch4a1ky1-C3_6cyc1oalkyl, -C(=0)-0H, -
NR.11aR1 lb and -NH-S(=0)2-C1-
4alkyl; and
C3_6cycloalkyl substituted by one or two substituents each independently
selected from the
group consisting of -CN, -OH, -0-C1_4alky1, -C(-0)-NH-Ci_4alkyl,
-NH-S(=0)2-Ci_4alkyl, and C1_4a1kyl optionally substituted with one
substituent selected from
the group consisting of OH, -0-C1_4a1kyl, -C(=0)-NH-Ci_4a1kyl and
-NH-S(=0)2-Ci_4alkyl.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R6 is selected from the group consisting of
Het3; -C(=0)-NH-R8;
C1_6alkyl substituted with one or two substituents each independently selected
from the group
consisting of Het3, Heft, Het6a, Het6b, Cy', _CN, -OH, -0-Ci_4a1ky1, -C(=0)-NH-
Ci_4a1ky1, -
C(=0)-NH-C1_4alkyl-C3_6cycloalkyl, -C(=0)-0H, -NR'laR1 lb and -NH-S(=0)2-
Ci_4a1ky1, and
C3_6cycloalkyl substituted by one or two substituents each independently
selected from the
group consisting of -CN, -OH, -0-Ci_4a1ky1, -C(=0)-NH-C1_4alkyl,
-NH-S(=0)2-Ci_4a1ky1, and Ci_4a1ky1 optionally substituted with one
substituent selected from
the group consisting of OH, -0-C1_4a1ky1, -C(=0)-NH-C1_4alkyl and
-NH-S(=0)2-Ci_4a1ky1.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R4 represents
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s'.s..
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R3 represents Het';
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is substituted on one nitrogen with R6; and wherein
said
heterocyclyl is optionally substituted on one or two carbon atoms with in
total one, two, three
or four halo sub stituents;
R6 is selected from the group consisting of C1_6alkyl optionally substituted
with one or two
sub stituents each independently selected from the group consisting of Het3,
Het4, Hee', and
Cy'.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
151 represents N;
R3 represents Het';
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is substituted on one nitrogen with R6; and wherein
said
heterocyclyl is optionally substituted on one or two carbon atoms with in
total one, two, three
or four halo sub stituents;
R6 is selected from the group consisting of Ci_6alkyl optionally substituted
with one or two
sub stituents each independently selected from the group consisting of Het3,
Het4, Hee', and
Cy'.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
represents N;
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Y1 represents -0-;
R" represents F;
R2 represents hydrogen;
Rzi represents isopropyl;
R3 represents Hal;
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is substituted on one nitrogen with R6; and wherein
said
heterocyclyl is optionally substituted on one or two carbon atoms with in
total one, two, three
or four halo sub stituents;
R6 is selected from the group consisting of Ci_6alkyl optionally substituted
with one or two
sub stituents each independently selected from the group consisting of Het3,
Het4, Het6a, and
Cy'.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
R3 represents Het';
Het' represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is substituted on one nitrogen with R6;
R6 represents C1_6alky1 substituted with one Het3.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
IJ1 represents N;
R3 represents Het';
Het' represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is substituted on one nitrogen with R6;
R6 represents C1_6alkyl substituted with one Het3.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
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pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein
151 represents N;
Y1 represents -0-,
Rib represents F;
R2 represents hydrogen;
R4 represents isopropyl;
R3 represents Het';
Heti represents a monocyclic C-linked 4- to 7-membered fully saturated
heterocyclyl
containing one N-atom;
wherein said heterocyclyl is substituted on one nitrogen with R6;
R6 represents C1_6a1kyl substituted with one Het3
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein the compounds of Formula
(I) are
restricted to compounds of Formula (I-y):
X
X
n3(g)n4
n1( )n2
Ri a
(I-y)
U2
U,
-1\1
wherein R3 is as defined for the compounds of Formula (I) or any subgroup
thereof as
mentioned in any of the other embodiments.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein the compounds of Formula
(I) are
restricted to compounds of Formula (I-y):
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4
2
X
n3( )n4
n1(8 )n2
R1 a
(I-y)
O2
wherein R3 represents Het'.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein the compounds of Formula
(I) are
restricted to compounds of Formula (I-y):
po3
4
12
X
n3(g)n4
n1( )n2
R1a
(I-y)
U 2
U
wherein R3 represents Cy2.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein the compounds of Formula
(I) are
restricted to compounds of Formula (I-z):
R3
R12 CYJ
0
r(j2
U
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wherein R3 is as defined for the compounds of Fon-nula (I) or any subgroup
thereof as
mentioned in any of the other embodiments.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein the compounds of Formula
(I) are
restricted to compounds of Formula (I-z):
R3
Rla CY-3
0 ylc, (I-Z)
U2
U
wherein R3 represents Het'.
In an embodiment, the present invention relates to those compounds of Formula
(I) and the
pharmaceutically acceptable salts, and the solvates thereof, or any subgroup
thereof as
mentioned in any of the other embodiments, wherein the compounds of Formula
(I) are
restricted to compounds of Formula (I-z):
R3
Rla
0 yl,õ 2 411 (1-4
U
wherein R3 represents Cy2.
In an embodiment, the present invention relates to a subgroup of Formula (I)
as defined in the
general reaction schemes.
In an embodiment the compound of Formula (I) is selected from the group
consisting of any
of the exemplified compounds,
tautomers and stereoisomeric forms thereof,
and the free bases, any pharmaceutically acceptable salts, and the solvates
thereof.
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All possible combinations of the above indicated embodiments are considered to
be embraced
within the scope of the invention.
METHODS FOR THE PREPARATION OF COMPOUNDS OF FORMULA (I)
In this section, as in all other sections unless the context indicates
otherwise, references to
Formula (I) also include all other sub-groups and examples thereof as defined
herein.
The general preparation of some typical examples of the compounds of Formula
(I) is described
hereunder and in the specific examples, and are generally prepared from
starting materials
which are either commercially available or prepared by standard synthetic
processes commonly
used by those skilled in the art of organic chemistry. The following schemes
are only meant to
represent examples of the invention and are in no way meant to be a limit of
the invention.
Alternatively, compounds of the present invention may also be prepared by
analogous reaction
protocols as described in the general schemes below, combined with standard
synthetic
processes commonly used by those skilled in the art.
The skilled person will realize that in the reactions described in the
Schemes, although this is
not always explicitly shown, it may be necessary to protect reactive
functional groups (for
example hydroxy, amino, or carboxy groups) where these are desired in the
final product, to
avoid their unwanted participation in the reactions. In general, conventional
protecting groups
can be used in accordance with standard practice. The protecting groups may be
removed at a
convenient subsequent stage using methods known from the art.
The skilled person will realize that in the reactions described in the
Schemes, it may be
advisable or necessary to perform the reaction under an inert atmosphere, such
as for example
under N2-gas atmosphere.
It will be apparent for the skilled person that it may be necessary to cool
the reaction mixture
before reaction work-up (refers to the series of manipulations required to
isolate and purify the
product(s) of a chemical reaction such as for example quenching, column
chromatography,
extraction).
The skilled person will realize that heating the reaction mixture under
stirring may enhance the
reaction outcome. In some reactions microwave heating may be used instead of
conventional
heating to shorten the overall reaction time.
The skilled person will realize that another sequence of the chemical
reactions shown in the
Schemes below, may also result in the desired compound of Formula (I).
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The skilled person will realize that intermediates and final compounds shown
in the Schemes
below may be further functionalized according to methods well-known by the
person skilled in
the art. The intermediates and compounds described herein can be isolated in
free form or as a
salt, or a solvate thereof. The intermediates and compounds described herein
may be
synthesized in the form of mixtures of tautomers and stereoisomeric forms that
can be separated
from one another following art-known resolution procedures.
SCHEME 1
In general, compounds of Formula (I) wherein Yl- is limited to Yla being -0-
or -NR5c-, hereby
named compounds of Formula (Ia), (Ib), (Ic), (Id), (Ie), can be prepared
according to the
following reaction Scheme 1. In Scheme 1, W1 represents fluoro, chloro, bromo
or iodo; all
other variables are defined according to the scope of the present invention.
R44 R3
R
i_ 4 R3 L.R3 R ---,xi.
X 1 1
12 X2 X2
X m1(S )m2 Rla
ml (5 )m2
m1(8 )m2 yl aH
WIn1( )n2
n1( )n2 Illa N
W1 1 N Rik) Ri a n1( N
)n2
v\& 1, u
IV
--N -W step 1 N ...-,....õ
W1 step 2 Rib N
-"'N W
II III (la)
R1,1=z3
12 step 3 HNR7aFeb
vv2mgcl_oikyi
x H 0-Ci_olkyl VI
VII
m1(8 )m2
V step 4 at 5 step
6
R1 a n1( N )n2
R4-õ..xi,R3 R4
3
yl_a _.., u
R,_R3
Y -1
N ,...,--2
X'I 2
X 12
X
Ft b I. '1\1 ml( X )m2 m1(8
)m2
ml( X )m2
(lb) )n2 RI,.
n1( )n2
Rth n1( N
1,1a n1( N )n2 N
-1-1 --U
410 NI_
,...i.... Rib 0 N ....õ-1, m 7b Rib
C1-4alkyl
(Id) (le)
(lc)
In Scheme 1, the following reaction conditions apply:
Step 1: at a suitable temperature such as ranged from room temperature to 90
C, in the presence
of a suitable base such as for example diisopropylethylamine or triethylamine
or sodium
carbonate, in a suitable solvent such as for example acetonitrile or di m
ethyl formami de or
dichloromethane;
Step 2: at a suitable temperature range from room temperature to 130 C, in
presence of a
suitable base such as for example cesium carbonate, in a suitable solvent such
as for example
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dimethylformamide or 1-methy1-2-pyrrolidinone;
Alternatively, at a suitable temperature such as for example room temperature,
in the presence
of a suitable deprotonating agent such as for example sodium hydride, in a
suitable solvent such
as for example dimethylsulfoxidc;
Alternatively, at a suitable temperature such as room temperature, in the
presence a suitable
base such as 1,8-Di azabi cyclo[5.4.0]undec-7-ene (DRU), in a suitable solvent
such as for
example tetrahydrofuran;
Step 3: at a suitable temperature such as room temperature, in the presence of
a suitable catalyst
such as palladium on charcoal (Pd/C), in a suitable solvent such as methanol,
under H2 pressure
such as for example from 1 to 3 bar, optionally in the presence of a base such
as triethylamine;
Alternatively, at a suitable temperature such as room temperature, in the
presence of a suitable
catalyst such as for example 1,1'-Bis(diphenylphosphino)ferrocene-
palladium(II)dichloride
dichloromethane complex, a suitable reducing agent such sodium borohydride, a
suitable base
such as for example /V,N,N',AP-tetramethylethylenediamine, in a suitable
solvent such as for
example tetrahydrofuran;
Step 4: at a suitable temperature range from 100 to 130 C, in presence of a
suitable base such
as for example cesium carbonate, in a suitable solvent such as for example
dimethylformamide
or 1-methy1-2-pyrrolidinone;
Step 5: at a suitable temperature range from 100 to 130 C, in presence of a
suitable base such
as for example cesium carbonate, in a suitable solvent such as for example
dimethylformamide
or 1-methy1-2-pyrrolidinone;
alternatively, at a suitable temperature ranged from 80 to 100 C, in presence
of a suitable
catalyst such as palladium acetate (Pd(OAc)2), in presence of a suitable
ligand such as for
example 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, in presence of a suitable
base such as
cesium carbonate, in a suitable solvent such as for example dioxane;
Step 6: at a suitable temperature from room temperature to 60 C, in presence
of a suitable
catalyst such as palladium acetate (Pd(OAc)2) or
Tris(dibenzylideneacetone)dipalladium(0)
(Pd2dba3), in presence or not of a suitable ligand such as for example
triphenylphosphine, in a
suitable solvent such as for example dioxane;
SCHEME 2
In general, compounds of Formula (I) wherein Y1 is limited to -CH2-, and R2 is
limited to W1,
hereby named compounds of Formula (If), can be prepared according to the
following reaction
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Scheme 2. In Scheme 2, all other variables are defined according to the scope
of the present
invention.
4 3 4
t_rcx1,R3
r,
-"X
12 12
X X
Ri a
n3( )n4 n3(
S )n4
CH2Zn Br
Rib
VIII
NI lb NI
VV .."1\1
W
step 1
In Scheme 2, the following reaction conditions apply:
Step 1: at a suitable temperature ranged from 60 C to 100 C, in presence of a
suitable catalyst
such as palladium acetate (Pd(OAc)2) or
Tris(dibenzylideneacetone)dipalladium(0)
(Pd2(dba)3) or Tetrakis(triphenylphosphine)palladium(0), in a suitable solvent
such as for
example tetrahydrofuran or dioxane.
The skilled person will realize that starting from compound (If), analogous
chemistry as
reported in steps 3, 4, 5 and 6 in scheme 1 could be performed.
SCHEME 3
In general, compounds of Formula (I) wherein Yl is limited to -CR5aR5b- and R2
is limited to
Wl, hereby named compounds of Formula (Ig), can be prepared according to the
following
reaction Scheme 3. In Scheme 3 at least one of R5a and R5b is other than
hydrogen. All other
variables are defined according to the scope of the present invention.
R4,1-R3 R4 R3
-X
X2 X2
Ri a R5a
n3( )n4
R5b n3( g )n4
n1( )n2 n1( )n2
Ri a
R5a R5b N
Rib Villa
,
I
wi
step 1 Rib
III (Ig)
In Scheme 3, the following reaction condition apply:
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Step 1: at a suitable temperature ranged from 80 C to 200 C, in presence of a
suitable catalyst
such as palladium acetate (Pd(OAc)2), in the presence of a suitable ligand
such as for example
triphenylphosphine or tricyclohexylphosphine, in a suitable solvent such as
for example
dioxane, preferably in sealed conditions, optionally under microwave
irradiation.
The skilled person will realize that starting from compound (Ig), analogous
chemistry as
reported in steps 3, 4, 5 and 6 in scheme 1 could be performed.
SCI IEME 4
In general, compounds of Formula (I) hereby named compounds of Formula (Ib)
can be
alternatively prepared according to the following reaction Scheme 4. In Scheme
4, PG1
represents a suitable protecting group, such as for example tert-
butyloxycarbonyl and LG1 is a
leaving group such as for example chloro, bromo, iodo or tosylate or mesylate;
all other
variables are defined as listed before or according to the scope of the
present invention.
PG1 PG1
2 PG1
1
PG1
1
)1/4'2 X X
n3( g )n4 n3( S )n4 R1' n3( X
)n4
Y1 alH
WI n1( )n2 n1( )n2
1:21a n1( )n2
N IX N N
w
H W I Rno IV
--==<"-L"----;(1-Y-LU
N j...,. , N ..,J, 1 I I
VV step 1 -'N¨ W step 2 R1''
II x XI
4
3
PG1 H
R--õõ.% iR
1 1
X2 0
)1(
X2
X2
L
n3( )n4 R4)..R3 X1113
n3( X )n4
n3( )n4
R1 n1( R
LG1
iRi, n1( N )n2
1 n1( S )n2
N)n2 1 2 '
YiY U R R XIllb
Y1Y1 u
Yi.YLI Rib N, ,j- Ri IV b '
N step 5 :lb
101 NC NC)
step 3 step 4
xii XIII (lb)
In Scheme 4, the following reaction conditions apply:
Step 1: at a suitable temperature such as ranged from room temperature to 90
C, in the presence
of a suitable base such as for example diisopropylethylamine or triethylamine
or sodium
carbonate, in a suitable solvent such as for example acetonitrile or
dimethylformamide or
dichloromethane;
Step 2: at a suitable temperature range from room temperature to 130 C, in
presence of a
suitable base such as for example cesium carbonate, in a suitable solvent such
as for example
dimethylformamide or 1-methyl-2-pyrrolidinone;
Alternatively, at a suitable temperature such as for example room temperature,
in the presence
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of a suitable deprotonating agent such as for example sodium hydride, in a
suitable solvent such
as for example dimethylsulfoxide;
Alternatively, at a suitable temperature such as room temperature, in the
presence a suitable
base such as 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), in a suitable solvent
such as for
example tetrahydrofuran;
Step 3: at a suitable temperature such as room temperature, in the presence of
a suitable catalyst
such as palladium on charcoal (Pd/C), in a suitable solvent such as methanol,
under H2 pressure
such as for example from 1 to 3 bar;
Alternatively, at a suitable temperature such as room temperature, in the
presence of a suitable
catalyst such as for example 1, r-Bis(diphenylphosphino)ferrocene-
palladium(II)dichloride
dichloromethane complex, a suitable reducing agent such sodium borohydride , a
suitable base
such as for example /V,N,N;N'-tetramethylethylenediamine, in a suitable
solvent such as for
example tetrahydrofuran;
Step 4: when PG' is tert-butyloxycarbonyl, at a suitable temperature range
such as for example
from 0 C to room temperature, in the presence of suitable cleavage
conditions, such as for
example an acid such as HCl or trifluoroacetic acid in a suitable solvent such
as acetonitrile or
DCM or methanol (Me0H);
Step 5: represents all type of reactions, such as for examples reductive
amination, nucleophilic
substitution, leading to final examples (lb);
The skilled person will realize that starting from intermediate XI, analogous
chemistry as
reported in steps 3, 4, 5 and 6 in scheme 1 could be performed.
S CI IEME 5
In general, compounds of Formula (I) wherein U is limited to N and Y1 is
limited to Ylb being
0, hereby named compounds of Formula (Iba) can be prepared according to the
following
reaction Scheme 5. In Scheme 5, PG' represents a suitable protecting group,
such as for
example tert-butyloxycarbonyl and 1A/2 a leaving group such as for example
chloro, tosylate or
mesylate; all other variables are defined according to the scope of the
present invention.
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Ria
lb
0 O-C1-4alkY1
Y.--- 0 0-C1_4alkyl Ri a 0 0 H
CI Rib IV yziX
yz j.....
YNji _________________________ a I N ___________ s I
NI
N'N-., "NI,--I
"NI
step 1 Rib 41 N step 2 Rib 0
N
II )(IV X\/
R4 3
R
X1
4
tõR3
X
i a
n3( )n4 12
X
R OH Ria
n3( )n4
ylb ,....1,_ VV2 n1( )n2
¨a- N'N. ¨3.- .`=N H Illa 1 n1(2
)n2
-Nri _) , R a N
R l b 401
step 3 step 4 R1 b 4111 'N step 5 Yi
IYN
XVI X\/II Rib 1
N I
"N,J
I411
P Gi
1 (lba)
0
YC2
n3( X )n4
R4)c3 Alia
step 6 ni( )n2
LGI
N IX step 8
12
H
R4,X.."'R3 )(Mb
PG1 H
I X2
X2
1_2
n3( )n4
n3( 2 )n2 )n4
N
Ria n1( )n2 Ri,
n1(
yz Ri b Si rei.....,,N
__________________________________________________________ r
N"N-)
-N1-1"N-)
step 7 Rib 140
XVIII >ix
In Scheme 5, the following reaction conditions apply:
Step 1: at a suitable temperature such as room temperature, in the presence of
a suitable base
such as for example potassium carbonate, in a suitable solvent such as for
example
dimethylformamide;
Step 2: at a suitable temperature such as room temperature, in presence of a
suitable base such
as lithium hydroxyde, in a suitable solvent such as for example a mixture of
tetrahydrofuran,
ethanol and water;
Step 3: at a suitable temperature such as room temperature, in the presence of
a
dibromoisocyanurate, in a suitable solvent such as dichloroethane;
Step 4: when W2 is chloro, at a suitable temperature range such as room
temperature, in the
presence of a chlorinating reagent such as oxalyl chlorine, in the presence of
a catalytic amount
of dimethylformamide, in the presence of a suitable base such as
triethylamine, in a suitable
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solvent such as dichloromethane;
When W2 is a trifluoroethoxy, at a suitable temperature such as 65 C, in the
presence of 2,2,2-
trifluoroethanol as solvent or not, suitable activating agents such as 1,3-
dibromo-1,3,5-
triazinane-2,4,6-trione, in the presence of molecular sieve;
Step 5: At a suitable temperature such as room temperature, in the presence of
a suitable base
such as for example triethylamine or 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU),
in a suitable
solvent such as for example dichloromethane or acetonitrile;
Step 6: At a suitable temperature such as room temperature, in the presence of
a suitable base
such as for example triethylamine or 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU),
in a suitable
solvent such as for example dichloromethane or acetonitrile;
Step 7: when PG' is tert-butyloxycarbonyl, at a suitable temperature range
such as for example
from 0 C to room temperature, in the presence of suitable cleavage
conditions, such as for
example an acid such as HC1 or trifluoroacetic acid in a suitable solvent such
as acetonitrile or
DCM or methanol (Me0H);
Step 8: represents all type of reactions, such as for examples reductive
amination, nucleophilic
substitution leading to final examples (Ib a).
SCHEME 6
In general, intermediates of formula Ma can be prepared according to the
following reaction
Scheme 5. In Scheme 5, PG2 represents a suitable protecting group, such as for
example
benzyloxycarbonyl; all other variables are defined according to the scope of
the present
invention or as defined in the previous schemes.
GPI GPI
R4---)LR3 XIlla R1R3
X
12 12 LGI
12
X X2 X 1
X
n3( )n4 n3( )n4 n3( )n4
XIllb n3(
)n4
n1( )n2 n1( )n2 3, n1( )n2
n1 (X )n2
1 2 1 2
step 1 GP step 2 GP
step 3
XX XXI XXII
Illa
Step 1: at a suitable temperature such as room temperature, in the presence of
benzyl
chloroformate, in the presence of a suitable base such as as for example
triethymaine, in a suitable
solvent such as for example dichloromethane;
Step 2: when PG' is tert-butyloxycarbonyl, at a suitable temperature range
such as for example
from 0 C to room temperature, in the presence of suitable cleavage
conditions, such as for
example an acid such as HC1 or trifluoroacetic acid in a suitable solvent such
as acetonitrile or
DCM or methanol (Me0H);
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Step 3: represents all type of reactions, such as for examples reductive
amination, nucleophilic
substitution leading to intermediate Ma.
SCHEME 7
In general, intermediates of formula XXVI can be prepared according to the
following reaction
Scheme 7. Variables are defined according to the scope of the present
invention or as defined
in the previous schemes.
Ria Rla
0
Ye
RIRS
1,1"- step 1 step 2 step 3 R,
XXIII XXIV XXV
XXVI
Step 1: at a suitable temperature such as 120 C, in the presence of a suitable
base such as for
example cesium carbonate, in a suitable solvent such as for example
dimethylacetamide;
Step 2: at a suitable temperature such as 0 C to room temperature, in the
presence of a suitable
oxidative agent such as for example urea hydrogen peroxide, in the presence of
a suitable
reagent such as trifluoroacetic anhydride, in a suitable solvent such as for
example
tett-ally drefuran,
Step 3: at a suitable temperature such as 0 C to room temperature, in the
presence of a suitable
chlorinated agent such as for example phosphoryl chloride, in the presence of
a suitable base
such as diisopropylethylamine, in a suitable solvent such as for example ethyl
acetate;
It will be clear for someone skilled in the art that starting from
intermediate XXVI, similar
chemistry as reported in Scheme 4 starting from intermediate II could be
performed.
SCHEME 8
In general, intermediates of formula XXVIII can be prepared according to the
following
reaction Scheme 8. Variables are defined according to the scope of the present
invention or as
defined in the previous schemes.
PG1
PG1 1
X2
X2 n3( g)n4
n3( g)n4
n1( )n2
n1( )n2
wi
IX
N
1 5 _, 2 1 5
W1 step 1 N W1
XXVII
XXVIII
Step 1: at a suitable temperature such as ranged from room temperature to 90
C, in the presence
of a suitable base such as for example diisopropylethylamine or triethylamine
or sodium
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carbonate, in a suitable solvent such as for example acetonitrile or
dimethylformamide or
dichloromethane;
It will be clear for someone skilled in the art that starting from
intermediate XXVIII, similar
chemistry as reported in Scheme 1 (ie steps 3, 4, 5 and 6) could be applied to
functionalize first
position 2. Then, from the obtained intermediate, similar chemistry as
reported in scheme 2 and
3 could be applied to functionalize position 5 with intermediates IV, VIII and
VIIa.
It will be appreciated that where appropriate functional groups exist,
compounds of various
formulae or any intermediates used in their preparation may be further
derivatized by one or
more standard synthetic methods employing condensation, substitution,
oxidation, reduction,
or cleavage reactions. Particular substitution approaches include conventional
alkylation,
aryl ation, heteroarylation, acylation, sulfonylation, halogenation,
nitration, formylation and
coupling procedures.
The compounds of Formula (I) may be synthesized in the form of racemic
mixtures of
enantiomers which can be separated from one another following art-known
resolution
procedures. The racemic compounds of Formula (I) containing a basic nitrogen
atom may be
converted into the corresponding di astereomeric salt forms by reaction with a
suitable chiral
acid. Said diastereomeric salt forms are subsequently separated, for example,
by selective or
fractional crystallization and the enantiomers are liberated therefrom by
alkali. An alternative
manner of separating the enantiomeric forms of the compounds of Formula (I)
involves liquid
chromatography using a chiral stationary phase. 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
stereospecifically.
In the preparation of compounds of the present invention, protection of remote
functionality
(e.g., primary or secondary amine) of intermediates may be necessary. The need
for such
protection will vary depending on the nature of the remote functionality and
the conditions of
the preparation methods. Suitable amino-protecting groups (NH-Pg) include
acetyl,
trifluoroacetyl, t-butoxycarbonyl (B oc),
benzyloxycarbonyl (CBz) and 9-
fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection is readily
determined by
one skilled in the art. For a general description of protecting groups and
their use, see T. W.
Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 4th ed.,
Wiley, Hoboken,
New Jersey, 2007.
PHARMACOLOGY
It has been found that the compounds of the present invention block the
interaction of menin
with MILL proteins and oncogenic MILL fusion proteins. Therefore the compounds
according
to the present invention and the pharmaceutical compositions comprising such
compounds
may be useful for the treatment or prevention, in particular treatment, of
diseases such as
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cancer, myelodysplastic syndrome (MDS) and diabetes.
In particular, the compounds according to the present invention and the
pharmaceutical
compositions thereof may be useful in the treatment or prevention of cancer.
According to
one embodiment, cancers that may benefit from a treatment with menin/MLL
inhibitors of the
invention comprise leukemias, myeloma or a solid tumor cancer (e.g. prostate
cancer, lung
cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma
and
glioblastoma, etc.). In some embodiments, the leukemias include acute
leukemias, chronic
leukemias, myeloid leukemias, myelogeneous leukemias, lymphoblastic leukemias,
lymphocytic leukemias, Acute myelogeneous leukemias (AML), Chronic myelogenous
leukemias (CML), Acute lymphoblastic leukemias (ALL), Chronic lymphocytic
leukemias
(CLL), T cell prolymphocytic leukemias (T-PLL), Large granular lymphocytic
leukemia,
Hairy cell leukemia (HCL), MILL-rearranged leukemias, MLL-PTD leukemias, MLL
amplified leukemias, MILL-positive leukemias, leukemias exphibiting 1-
10XIMEISI gene
expression signatures etc.
Hence, the invention relates to compounds of Formula (I), the tautomers and
the
stereoisomeric forms thereof, and the pharmaceutically acceptable salts, and
the solvates
thereof, for use as a medicament.
The invention also relates to the use of a compound of Formula (I), a tautomer
or a
stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a
solvate thereof, or a
pharmaceutical composition according to the invention, for the manufacture of
a medicament.
The present invention also relates to a compound of Formula (I), a tautomer or
a
stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a
solvate thereof, or a
pharmaceutical composition according to the invention, for use in the
treatment, prevention,
amelioration, control or reduction of the risk of disorders associated with
the interaction of
menin with MILL proteins and oncogenic MLL fusion proteins in a mammal,
including a
human, the treatment or prevention of which is affected or facilitated by
blocking the
interaction of menin with MLL proteins and oncogenic MILL fusion proteins.
Also, the present invention relates to the use of a compound of Formula (I), a
tautomer or a
stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a
solvate thereof, or a
pharmaceutical composition according to the invention, for the manufacture of
a medicament
for treating, preventing, ameliorating, controlling or reducing the risk of
disorders associated
with the interaction of menin with MLL proteins and oncogenic MILL fusion
proteins in a
mammal, including a human, the treatment or prevention of which is affected or
facilitated by
blocking the interaction of menin with MILL proteins and oncogenic MILL fusion
proteins.
The invention also relates to a compound of Formula (I), a tautomer or a
stereoisomeric form
thereof, or a pharmaceutically acceptable salt, or a solvate thereof, for use
in the treatment or
prevention of any one of the diseases mentioned hereinbefore.
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The invention also relates to a compound of Formula (I), a tautomer or a
stereoisomeric form
thereof, or a pharmaceutically acceptable salt, or a solvate thereof, for use
in treating or
preventing any one of the diseases mentioned hereinbefore.
The invention also relates to the use of a compound of Formula (I), a tautomer
or a
stereoisomeric form thereof, or a pharmaceutically acceptable salt, or a
solvate thereof, for the
manufacture of a medicament for the treatment or prevention of any one of the
disease
conditions mentioned hereinbefore
The compounds of the present invention can be administered to mammals,
preferably humans,
for the treatment or prevention of any one of the diseases mentioned
hereinbefore
In view of the utility of the compounds of Formula (I), the tautomers and the
stereoisomeric
forms thereof, and the pharmaceutically acceptable salts, and the solvates
thereof, there is
provided a method of treating warm-blooded animals, including humans,
suffering from any
one of the diseases mentioned hereinbefore.
Said method comprises the administration, i.e. the systemic or topical
administration, of a
therapeutically effective amount of a compound of Formula (I), a tautomer or a
stereoisomeric
form thereof, or a pharmaceutically acceptable salt, or a solvate thereof, to
warm-blooded
animals, including humans.
Therefore, the invention also relates to a method for the treatment or
prevention of any one of
the diseases mentioned hereinbefore comprising administering a therapeutically
effective
amount of compound according to the invention to a patient in need thereof
One skilled in the art will recognize that a therapeutically effective amount
of the compounds
of the present invention is the amount sufficient to have therapeutic activity
and that this
amount varies inter alias, depending on the type of disease, the concentration
of the
compound in the therapeutic formulation, and the condition of the patient. An
effective
therapeutic daily amount would be from about 0.005 mg/kg to 100 mg/kg. The
amount of a
compound according to the present invention, also referred to herein as the
active ingredient,
which is required to achieve a therapeutically effect may vary on case-by-case
basis, for
example with the particular compound, the route of administration, the age and
condition of
the recipient, and the particular disorder or disease being treated. A method
of treatment may
also include administering the active ingredient on a regimen of between one
and four intakes
per day. In these methods of treatment the compounds according to the
invention are
preferably formulated prior to administration.
The present invention also provides compositions for preventing or treating
the disorders
referred to herein. Said compositions comprising a therapeutically effective
amount of a
compound of Formula (I), a tautomer or a stereoisomeric form thereof, or a
pharmaceutically
acceptable salt, or a solvate thereof, and a pharmaceutically acceptable
carrier or diluent.
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While it is possible for the active ingredient to be administered alone, it is
preferable to
present it as a pharmaceutical composition. Accordingly, the present invention
further
provides a pharmaceutical composition comprising a compound according to the
present
invention, together with a pharmaceutically acceptable carrier or diluent. The
carrier or diluent
must be "acceptable" in the sense of being compatible with the other
ingredients of the
composition and not deleterious to the recipients thereof.
The pharmaceutical compositions may be prepared by any methods well known in
the art of
pharmacy, for example, using methods such as those described in Gennaro et al.
Remington's
Pharmaceutical Sciences (18th ed., Mack Publishing Company, 1990, see
especially Part 8 :
Pharmaceutical preparations and their Manufacture).
The compounds of the present invention may be administered alone or in
combination with
one or more additional therapeutic agents. Combination therapy includes
administration of a
single pharmaceutical dosage formulation which contains a compound according
to the
present invention and one or more additional therapeutic agents, as well as
administration of
the compound according to the present invention and each additional
therapeutic agent in its
own separate pharmaceutical dosage formulation.
Therefore, an embodiment of the present invention relates to a product
containing as first
active ingredient a compound according to the invention and as further active
ingredient one
or more anticancer agent, as a combined preparation for simultaneous, separate
or sequential
use in the treatment of patients suffering from cancer.
The one or more other medicinal agents and the compound according to the
present invention
may be administered simultaneously (e.g. in separate or unitary compositions)
or sequentially
in either order. In the latter case, the two or more compounds will be
administered within a
period and in an amount and manner that is sufficient to ensure that an
advantageous or
synergistic effect is achieved. It will be appreciated that the preferred
method and order of
administration and the respective dosage amounts and regimes for each
component of the
combination will depend on the particular other medicinal agent and compound
of the present
invention being administered, their route of administration, the particular
condition, in
particular tumour, being treated and the particular host being treated.
The following examples further illustrate the present invention.
EXAMP1,kS
Several methods for preparing the compounds of this invention are illustrated
in the following
examples. Unless otherwise noted, all starting materials were obtained from
commercial
suppliers and used without further purification, or alternatively can be
synthesized by a skilled
person by using well-known methods.
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As understood by a person skilled in the art, compounds synthesized using the
protocols as
indicated may exist as a solvate e.g. hydrate, and/or contain residual solvent
or minor impurities.
Compounds isolated as a salt form, may be integer stoichiometric i.e. mono- or
di-salts, or of
intermediate stoichiometry. When an intermediate or compound in the
experimental part below
is indicated as 'HCl salt' without indication of the number of equivalents of
HC1, this means
that the number of equivalents of HC1 was not determined.
The stereochemical configuration for centers in some compounds may be
designated "R" or "S"
when the mixture(s) was separated; for some compounds, the stereochemical
configuration at
indicated centers has been designated as "*R" or "*S" when the absolute
stcreochemistry is
undetermined (even if the bonds are drawn stereo specifically) although the
compound itself
has been isolated as a single stereoisomer and is enantiomerically pure.
For example, it will be clear that Compound 3
0
N *R
NO
ON
is
d_112---cR
N s
N N
or
0
,11\1 N
I
N
F
The paragraphs above about stereochemical configurations, also apply to
intermediates.
The term "enantiomerically pure" as used herein means that the product
contains at least 80%
by weight of one enantiomer and 20% by weight or less of the other enantiomer.
Preferably the
product contains at least 90% by weight of one enantiomer and 10% by weight or
less of the
other enantiomer. In the most preferred embodiment the term "enantiomerically
pure" means
that the composition contains at least 99% by weight of one enantiomer and 1%
or less of the
other enantiomer.
A skilled person will realize that, even where not mentioned explicitly in the
experimental
protocols below, typically after a column chromatography purification, the
desired fractions
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were collected and the solvent was evaporated.
In case no stereochemistry is indicated, this means it is a mixture of
stereoisomers, unless
otherwise is indicated or is clear from the context.
When a stereocenter is indicated with `RS' this means that a racemic mixture
was obtained at
the indicated centre, unless otherwise indicated.
A skilled person will understand that when Intermediates or Compounds are
reported in Tables,
the synthetic methodology from the indicated starting material to desired
Intermediate/Compound might go over one or more reaction steps.
When two enantiomers, diastereomers or isomers are present in the same cell of
one of the
tables below, a skilled person will understand that these Intermediates or
Compounds were
separated from each other by using a suitable chromatographic method e.g. SFC
or reversed
phase separation.
Preparation of intermediates
For intermediates that were used in a next reaction step as a crude or as a
partially purified
intermediate, in some cases no mol amounts are mentioned for such intermediate
in the next
reaction step or alternatively estimated mol amounts or theoretical mol
amounts for such
intermediate in the next reaction step are indicated in the reaction protocols
described below.
Hereinafter, the terms : `ACNI or `MeCN' means acetonitrile, `DCM' means
dichloromethane,
`DIPEA or DMA' means N,N-diisopropylethylamine, 'h' means hours(s), 'min'
means
minute(s), `DMF' means 107,N-dimethylformamide, 'TEA' or 'Et3N' means triethyl
amine,
'Et0Ac' or 'EA' means ethyl acetate, 'THE' means tetrahydrofuran; `HPLC' means
High-
performance Liquid Chromatography, Prep-I-IPLC ' means preparative I-TPLC;
MeOIT means
methanol, `NMIR' means Nuclear Magnetic Resonance, or
`RT' means room temperature,
SFC' means supercritical fluid chromatography, `q.s.' means quantum satis,
'DMS0' means
dimethylsulfoxide, `Pd/C'or "Pd/C (10%)" means palladium on carbon, 'atm'
means
atmosphere, 'cc' means enantiomeric excess, 'PE' means petroleum ether,
`NaBH(OAc)3'
means sodium triacetoxyborohydride, `TFA' means trifluoroacetic acid, `DCE'
means
dichloroethane, and 'DMA' means N,N-dimethylacetamide; "IPA" means isopropyl
alcohol;
"iPrNH2" means isopropylamine; NH4OH means ammonium hydroxide; "Pd(OH)2 means
palladium hydroxide; DBU means 1,8-diazabicyclo[5.4.01undec-7-ene; Cbz" means
benzoylcarbonyl; NaBH3CN means sodium cyanoborohydride; NaBH4 means sodium
borohydride; tic means thin-layer chromatography; FCC means Flash Column
Chromatography; HATU means 1-[Bi s(dimethyl amino)methyl ene]-1H-1,2,3 -tri
azol o [4, 5-
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b]pyridinium 3 -oxid hexafluorophosphate, N-RDimethyl amino)-1H-1,2,3 -triazol
o-[4, 5-
b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide;
EDCI
means N-(3 -Dimethylaminopropy1)-N'-ethyl carbodiimide hydrochloride, `HOBT'
or `HOBt'
means 1-Hy droxyb enzotri azol e hydrate; TMEDA
means N,N,N' ,AP -
Tetram ethyl ethyl enedi amine; Pd(dppf)C12.DCM means
[1,1'-
Bis(diphenylphosphino)ferroceneldichloropalladium(II), complex with
dichloromethane;
"Ni(acac)2" means Nickel(II) acetylacetonate; "Zn" means Zinc; "MS" means
molecular sieve;
"Boc20" means di-tert-butyl decarbonate; "Ar" means argon; "FA" means formic
acid; "CC"
means column chromatography; "T3P" means propyl phosphonic anhydride.
A. Preparation of the intermediates
Example Al
Preparation of intermediate 1
N,Boc
RS
0 0
A mixture of 2,6-diazaspiro[3.3]heptane-2-carboxylic acid, phenylmethyl ester
(1.084g. 4.667
mmol), tert-butyl 3 -i sobutyryl azeti di ne-1 - carb oxyl ate (1.3 g, 5.6
mmol), sodium
cyanoborohydride (1.5 g, 23.33 mmol) and acetic acid (267 pL, 4.67 mmol) in
methanol (50
mL) was stirred at 50 C overnight. The mixture was gathered with another
reaction performed
on 100 mg of 2,6-diazaspiro[3.3]heptane-2-carboxylic acid, phenylmethyl ester
and poured
onto 10% aqueous solution of K2CO3. The resulting mixture was extracted with
DCM. The
organic layer was decanted, washed with water, dried over MgSO4, filtered and
evaporated to
dryness. The residue was purified by chromatography over silica gel (irregular
SiOH, 40g;
mobile phase: gradient from 0% NH4OH, 0% Me0H, 100% DCM to 03% NH4OH, 3% Me0H,
97% DCM). The pure fractions were collected and evaporated to dryness. The
resulting residue
was purified a second time by chromatography over silica gel (irregular SIOH,
40g; mobile
phase: gradient from 40% Et0Ac, 60% heptane to 60% Et0Ac, 40% heptane). The
pure
fractions were collected and evaporated to dryness yielding 1.58 g of
intermediate 1 (70% yield).
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Preparation of intermediate 2
N H
RS
0'.0'
A mixture of intermediate 1 (500 mg; 1.127 mmol) and TF A (1.5 mL) in DCM (5
mL) was
stirred at room temperature overnight. The reaction mixture was diluted with
ACN and
evaporated to dryness (twice). The residue was dissolved in DCM and basified
with 15%
aqueous solution of NH4OH. The organic layer was washed again with 15% aqueous
solution
of NH4OH, then, with water, filtered over Chromabond and evaporated to
dryness yielding
330 mg of intermediate 2 (85%) which was directly engaged in the next step
without any further
purification.
Preparation of intermediate 3
N
0
RS
X
0 0
Acetic acid (55 [iL; 0,96 mmol) was added at room temperature to a solution of
intermediate 2
(330 mg; 0.96 mmol) and oxetane-3-carbaldehyde (132 jut; 1.92 mmol) in THF (12
mL). The
mixture was stirred at rt for overnight then NaBH(OAc)3 (611 mg; 2.88 mmol)
was added
portionwise. The mixture was stirred at rt for 3 hours. The reaction mixture
was partitioned
between aqueous 10% K2CO3 and Et0Ac. The layers were separated and the aqueous
layer was
extracted once with DCM. The organic layers were mixed, dried over MgSO4 and
evaporated
to dryness. The residue was purified by chromatography over silica gel
(irregular SiOH,
10g-F24g; mobile phase: gradient from 0.5% NH4OH, 5% Me0H, 95% DCM to 1%
NH4OH,
10% Me0H, 90% DCM). The pure fractions were collected and evaporated to
dryness yielding
264 mg of intermediate 3 (66% yield).
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Preparation of intermediate 4
NO
RS
A mixture of intermediate 3 (264 mg; 0.638 mmol) and Pd/C (10%) (68 mg; 0.0638
mmol) in
ethanol (10 mL) was hydrogenated under 3 bars of H2 for 2 hours. Pd/C (10%)
was removed
by filtration over celite and the solvent was evaporated to dryness yielding
173 mg of
intermediate 4 (97% yield).
Example A2
Preparation of intermediate 5
Boc
RS
0
In a round bottom flask, 2,6-diazaspiro[3.4]octane-6-carboxylic acid,
phenylmethyl ester (500
mg; 2.03 mmol), tert-butyl 3-isobutyrylazetidine-1-carboxylate (553.7 mg; 2.43
mmol), sodium
cyanoborohydride (382.7 mg; 6.09 mmol) and acetic acid (0.116 mL; 2.03 mmol)
were diluted
in Me0H. Then, the reaction mixture was heated overnight at 50 C and cooled
down to room
temperature. Carefully, a saturated solution of Na,HCO3 was added until pH >
9. The resulting
mixture was extracted with DCM. The organic layer was decanted, washed with
water, dried
over MgSO4, filtered and evaporated to dryness. The residue was purified by
chromatography
over silica gel (irregular SiOH, 40g; mobile phase: gradient from 0% NH4OH, 0%
Me0H, 100%
DCM to 0.3% NI-140H, 3% Me0H, 97% DCM). The pure fractions were collected and
evaporated to dryness to afford 700 mg of intermediate 5 (75% yield)
Preparation of intermediate 6
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NH
RS
0
In a round bottom flask, at 0 C, TFA (2.34 mL, 30.59 mmol) was added to
intermediate 5 (700
mg, 1.53 mmol) in DCM (33.7 mL). Then, the reaction was warmed to room
temperature and
the reaction mixture was stirred overnight at room temperature The residue was
dissolved in 4
mL of water. Then, the solution was basified with a solution of NaOH 1M (12
mL) until pH=8-
9. After stirring for 10 min at room temperature, the resulting mixture was
extracted with
dichloromethane (3 x 30 mL). The combined organic layers were washed with
brine (1 x 50
mL), dried over MgSO4, filtered and evaporated till dryness to give 482 mg of
intermediate 6
which was directly engaged in the next step without any further treatment.
Preparation of intermediate 7
N
RS
0
Acetic acid (119 ttL; 207 mmol) was added at room temperature to a solution of
intermediate
6 (482 mg; 1.34 mmol) and oxetane-3-carbaldehyde (188 pL; 2.72 mmol) in THF
(20 mL). The
mixture was stirred at rt for 4h then NaBH(OAc)3 (870 mg; 4.1 mmol) was added
portionwise.
The mixture was stirred at room temperature for 2 hours. The reaction mixture
was poured into
ice water, basified with an aqueous solution of K2CO3 10% and Et0Ac was added.
The organic
layer was separated, washed with brine, dried over MgSO4, filtered and
evaporated till dryness
to give 438 mg of an intermediate residue. The residue (438 mg) was purified
by silica gel
chromatography (Stationary phase: irregular SiOH 15-401.tm 24g Mobile phase:
Gradient from
97% DCM, 3% Me0H (+10% NH4OH) to 90% DCM, 10% Me0H (+10% N1140H)). The
fractions containing the product were mixed and concentrated to give 127 mg of
intermediate
7 (22% yield).
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Preparation of intermediate 8
RS
NH
A mixture of intermediate 7 (63 mg; 0.147 mmol), Pd(OH)2 (21 mg; 0.174 mmol)
in Me0H (3
mL) and THF (0.5 mL) was hydrogenated under atmospheric pressure overnight.
The catalyst
was removed by filtration through a pad of celite', washed with Me0H and the
filtrate was
evaporated to give 35 mg of intermediate 8 (81% yield).
Example A3
Preparation of intermediate 9
0
To the mixture of 5-fluoro-2-methoxybenzoic acid (8.00 g, 47.0 mmol) and N-
ethylpropan-2-
amine (8.19 g, 94.0 mmol) in dry DCM (150 mL) cooled at 0 C, were slowly
added HATU
(21.5 g, 56.5 mmol) and DIEA (9.10 g, 70.4 mmol) in portions. The resulting
mixture was
slowly warmed to RT and stirred for 8 h. The organic layer was washed with
water (20 mL x
3) and dried over anhydrous Na2SO4. After filtration, the solvent was removed
under reduced
pressure and the crude product was purified by FCC (Et0Ac/PE = 0% to 20% of
Et0Ac) to
afford intermediate 9 (12.0 g, 96% yield) as a white solid.
The following intermediate was synthesized by an analogous method as described
above for
the preparation of intermediate 9
Int. No. Structure Starting Materials
0 5 -fluoro-2-m ethoxyb enzoi c
acid,
0 dii sopropyl amine
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Preparation of intermediate 11 (Method A)
F
To the solution of intermediate 9(12.0 g, 50.1 mmol) in dry DCM (100 mL)
cooled at -78 C
was slowly added BBr3 (14.4 mL, 152 mmol) and the resulting mixture was slowly
warmed to
RT and stirred for 8 h. The mixture was cooled to -78 C again and Me0H (5 mL)
was added
dropwise to quench the reaction. The resulting mixture was slowly warmed to RT
and the pH
value was adjusted to about 8 by adding a saturated solution of NaHCO3. The
aqueous layer
was extracted by DCM (50 mL x 3) and the combined organic layers were dried
over anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give the crude
product which was
purified by FCC (Et0Ac/PE = 0% to 20% of Et0Ac) to afford intermediate 11(9.0
g, 78%
yield) as a white solid.
Alternative preparation of intermediate 11 (Method B)
0
OH
A solution of 5-fluorosalicylic acid (30.0 g, 192.2 mmol) in thionyl chloride
(200 mL) was
stirred for 5 hours at 80 C. Then, the resulting mixture was concentrated
under reduced
pressure to give the acyl chloride. To a stirred solution of N-ethylpropan-2-
amine (33.5 g,
384.3 mmol) and triethyl amine (58.3 g, 576.5 mmol) in dichloromethane (200
mL) was added
a solution of acyl chloride in dichloromethane (100 mL) dropwise at 0 'C.
After stirring
overnight at room temperature, the resulting mixture was concentrated under
reduced pressure.
The crude product was dissolved in methanol (300 mL). Then, a solution of
sodium hydroxide
(20 g) in water (100 mL) was added. After stirring for 1 hour at room
temperature, the resulting
mixture was diluted with water (100 mL) and concentrated under reduced
pressure to remove
the excess methanol, adjusted to pH value 4 and extracted with ethyl acetate
(2 x 150 mL).
The combined organic layer was concentrated under reduced pressure. The
residue was
purified by silica gel column chromatography, eluted with (EA/PE, 16.3:83.7)
to afford 29.4
g (66% yield) of intermediate 11 as an off-white solid.
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The following intermediate was synthesized by an analogous method as described
above for
intermediate 11 (Method A)
Int. No. Structure Starting Materials
N 0
12 intermediate 10
F
The following intermediate was synthesized by an analogous method as described
above for
intermediate 11 (Method B)
Int. No. Structure Starting Materials
0
81 OH 5-fluorosalicylic acid
Example A4
Preparation of intermediate 13
,Boc
CI
*LI N
N ,NCI
To the solution of 3,5,6-trichloro-1,2,4-triazine (10.0 g, 54.2 mmol) and TEA
(15.2 mL, 109
mmol) in DCM (100 mL) cooled at 0 C was added tert-butyl 2,6-
diazaspiro[3.4]octane-2-
carboxylate (9.21 g, 43.4 mmol) and the mixture was warmed to RT and stirred
for 1 h. The
mixture was diluted with water (20 mL) and extracted with DCM (30 mL x 3). The
combined
organic layers were washed with brine, dried over Na2SO4, filtered and
concentrated under
reduced pressure to give the crude product which was purified by FCC on silica
gel (Mobile
phase A: PE; Mobile phase B: Et0Ac, eluent from 0-25% Mobile phase B) to
afford
intermediate 13 (12.0 g, 58% yield) as a yellow solid.
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Preparation of intermediate 14
Boc
0
Oy-LN
N,N-õ-1,CI
The mixture of intermediate 13 (12.0 g, 33.3 mmol), intermediate 11(7.5 g,
33.3 mmol) and
DB U (6.1 g, 40.1 mmol) in TI-IF (120 mL) was stirred at 25 C for 8 h. The
mixture was diluted
with water (30 mL) and extracted with DCM (30 mL x 3). The combined organic
layers were
washed with brine, dried over Na2SO4, filtered and concentrated under reduced
pressure to give
the crude product which was purified by FCC on silica gel (Mobile phase A: PE;
Mobile phase
B: Et0Ac, eluent from 0-25% Mobile phase B) to afford intermediate 14 (14.0 g,
73% yield)
as a green solid.
The following intermediates was synthesized by an analogous method as
described above for
intermediate 14
Int. No. Structure Starting Materials
Boc
0 intermediate 12,
intermediate 13
0 N
N,NCI
Preparation of intermediate 16
,Boc
0
0 yk\ N
I
Method A:
To the mixture intermediate 14 (20 g, 36.4 mmol), NaBH4 (2.48 g, 65.7 mmol)
and TMEDA
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(8.54 g, 73.5 mmol) in THF (500 mL) was added Pd(dppf)C12=DCM (1.70 g, 2.08
mmol) under
N2 atmosphere. After addition, the reaction mixture was stirred at 25 C for
14 h. The reaction
mixture was filtered, and the filtrate was concentrated, the residue was
purified by FCC on
silica gel (eluent with Et0Ac) to afford intermediate 16 (15 g, 74% yield) as
brown solid.
Method B:
To the solution of intermediate 14 (22.0 g, 40.1 mmol), TEA (15 mL) in Me0H
(100 mL) was
added Pd/C (wet, 5.0 g, 10%) The resulting mixture was stirred under H2
atmosphere (30 psi)
at 25 C for 8hr. The reaction mixture was filtered through a celite pad and
the filtrate was
concentrated in vacuo to afford intermediate 16 (25.0 g, crude), which was
used directly in next
step without further purification.
The following intermediate was synthesized by an analogous method described
above for
intermediate 16
Int. No. Structure Starting Material
Conditions
,Boc
Pd/C, H2,
17 intermediate 15
TEA, Me0H
I I
F N
Preparation of intermediate 18
NH
NO
ON
1
NN
To the solution of intermediate 16 (300 mg, 0.583 mmol) in DCM (5 mL) was
added TFA (0.5
mL, 6.4 mmol) and the resulting mixture was stirred at RT for 3 h. Then, 10%
NaOH (5 mL)
solution was slowly added into the mixture to adjust the pH value to about 12
and the resulting
mixture was extracted with DCM (10 mL x 3). The combined organic layers were
dried over
anhydrous Na2SO4, filtered, and concentrated in vacno to afford intermediate
18 (220 mg, 90%
yield) as a white solid.
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The following intermediate was synthesized by an analogous method described
above for
intermediate 18
Int. No. Structure Starting Material
NH
19 0
intermediate 17
N
I
NN
Example A5
Preparation of intermediate 20:
\ /
NH
O¨N H
0 Boc
To a solution of cis-3- [[(1,1-dimethylethoxy)carbonyl]amino]-
cyclobutanecarboxylic acid
(10.0 g, 46.5 mmol) in DIN,IF (100 mL) was added HOBt (8.15 g, 60.3 mmol),
EDCI (11.6 g,
60.5 mmol) and DIEA (30.0 mL, 182 mmol, 0.782 g/mL) at 0 C. Then, N,0-
dimethylhydroxylamine hydrochloride (5.90 g, 60.5 mmol) was added at 0 C. The
mixture was
stirred at room temperature for 16 hours. The mixture was diluted with ethyl
acetate (500 mL).
The mixture was washed with 1 M HC1 (150 mL), saturated NaHCO3 (100 mL x 2)
and brine
(300 mL x 3), dried over Na2SO4, filtered and concentrated under reduced
pressure to give
intermediate 20 (11.0 g, crude) as a white solid, which was used in the next
step without further
purification.
Preparation of intermediate 21:
NH
0 Boc
To a solution of intermediate 20 (11.0 g, 6.97 mmol) in TEM (100 mL) was added
isopropylmagnesium chloride (64.0 mL, 128 mmol, 2M in THF) dropwise at 0 C
under N2
atmosphere. The mixture was stirred at room temperature for 12 hours under N2
atmosphere.
The mixture was quenched with saturated NH4CI (100 mL). The mixture was
filtered through
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a pad of Celite and the filtrate was concentrated under reduced pressure. The
mixture was
extracted with ethyl acetate (200 mL x 2). The combined organic layers were
washed with brine
(200 mL x 2), dried over Na2SO4, filtered and concentrated under reduced
pressure. The crude
product was purified by flash column chromatography over silica gel (eluent:
petroleum ether:
ethyl acetate from 1:0 to 5:1, to yield intermediate 21(6.30 g) as a white
solid.
Example A6
Preparation of intermediate 22:
N HBoc
RS
N 0
0 ,L.N
I
N,N-;)
Under N2, at rt, to a mixture of intermediatel8 (1 g, 2.41 mmol), intermediate
21(873 mg, 3.62
mmol), acetic acid (276 uL, 4.83 mmol) in Me0H (50 mL) was added NaBH3CN (455
mg,
7.24 mmol). Then, the reaction was heated at 50 C overnight. The reaction
mixture was cooled
to rt, poured into ice water, basified with a saturated solution of NaHCO3 and
DCM was added.
The organic layer was separated, washed with brine, dried over MgSO4, filtered
and evaporated
till dryness. The crude was purified by silica gel chromatography (Stationary
phase: irregular
SiOH 15-40 m 40g, Mobile phase: Gradient from 0% NH4OH, 100% DCM, 0% Me0H to
0.1%
NH4OH, 95% DCM, 5% Me0H). The fraction containing the product were mixed and
concentrated to afford 1.37g (89% yield) of intermediate 22.
Example A7
Preparation of intermediate 23:
\ /
0¨N
To a solution of 3,3-dimethoxycyclobutanecarboxylic acid (12.0 g, 75 mmol) in
DCM (145 mL)
was added T3P (100 mL, 168 mmol, 50% in Et0Ac) and DIEA (64 mL, 372 mmol) at 0
C.
Then N,O-dimethylhydroxylamine hydrochloride (8.8 g, 89.5 mmol) was added at 0
C. The
mixture was stirred at room temperature for 16 hours. The mixture was poured
onto a saturated
solution NaHCO3 and Et0Ac was added. The organic layer was separated, washed
with brine,
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dried over MgSO4, filtered and concentrated under reduced pressure to give
intermediate 23
(16.0 g, crude) which was used in the next step without further purification.
Preparation of intermediate 24:
0-
0 0'
The reaction was performed twice on 15.7 g of intermediate 23 and respective
reaction media
were mixed for the work-up and purification. To a solution of intermediate 23
(15.7 g, 77.7
mmol) in THE (420 mL) was added isopropylmagnesium chloride (178.5 mL, 232
mmol, 2M
in THF) dropwise at 0 C under N2 atmosphere. The reaction mixtures were
stirred at room
temperature for 12 hours under N2 atmosphere and then, poured onto ice-water
and a 10%
aqueous solution of NH4C1. The mixture obtained was combined with the mixture
obtained
from the second reaction, and the combined mixture was extracted with Et0Ac.
The combined
organic layers were washed with brine, dried over MgSO4, filtered and
concentrated under
reduced pressure. The crude product was purified by flash column
chromatography over silica
gel (mobile phase: Heptane: Et0Ac 9:1). The pure fractions were collected and
evaporated to
dryness yielding 22 g (76% yield) of intermediate 24 as a colourless oil.
Example A8
Preparation of intermediate 25
o
0
ON
I
A mixture of intermediate 18 (10 g, 24.13 mmol), intermediate 24 (4.94 g,
26.54 mmol) and
acetic acid (1.5 mL, 26.54 mmol) in Me0H (80 mL) was stirred at room
temperature for 20
min. Then, NaBH3CN (1.82 g, 28.95 mmol) was added and the mixture was stirred
at 50 C
overnight. The reaction solution was poured into ice water and extracted with
DCM. The
organic layer was washed with water and brine, then dried over Na2SO4,
filtered and
concentrated under reduced pressure. The residue was purified by flash
chromatography on
silica gel (Mobile phase A: PE; Mobile phase B: Et0Ac, eluent from 0-100%
Et0Ac) to give
9.21 g (64% yield) of intermediate 25 as a light yellow solid.
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Preparation of intermediate 26
¨/
N R
N
I
N., -,;)
and intermediate
27
S
0
0
N,N
Intermediate 25 (9.2g) was purified via chiral SFC (Stationary phase:
C+HIRALPAK AD-H
5pm 250*21.2mm, Mobile phase: 83% CO2, 17% mixture of Et0H/ACN 80/20
v/v(+0.3%iPrNH2)). The fractions containing the products were mixed and
concentrated to
afford 4.07g (44% yield) of intermediate 26 and 4.06 g (44% yield) of
intermediate 27 and 273
mg of a residual fraction of intermediate 25.
Method A for intermediate 27:
To a solution of intermediate 35 (2.24 g, 3.618 mmol) in methanol (45 mL) was
added
palladium on activated carbon (10% palladium) (635 mg, 0.597 mmol). Then, the
mixture was
stirred at room temperature for 5 hours under the hydrogen. The mixture was
diluted with
methanol, filtered through a pad of Celite and the filtrate was evaporated
under reduced
pressure. The residue was dissolved with ethyl acetate, washed with sodium
hydroxide solution
(1M in water) and brine. The organic layer was dried over anhydrous sodium
sulfate, evaporated
under reduced pressure to give 1.4 g (62% yield) of intermediate 27 as a
yellow solid.
Method B for intermediate 27:
A mixture of intermediate 35 (1.44 g; 2.33 mmol) and TMEDA (0.54 mL; 3.63
mmol) in dry
THE (55 mL) was degassed by N2 bubbling. Then, Pd(dppf)C12.DCM (216 mg; 0.26
mmol)
and sodium borohydride (144 mg; 3.81 mmol) were added. The reaction mixture
was stirred at
50 C overnight in a sealed glassware. The solution was cooled, poured out into
cooled water.
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Et0Ac was added and the mixture was filtered through a celite . The product
was extracted
with Et0Ac and the organic layer was dried over MgSO4, filtered and evaporated
to dryness.
The crude residue (1.7g) was purified by silica gel chromatography (Stationary
phase: irregular
SiOH 40 p.m 40 g, Mobile phase: Gradient from 100% DCM, 0% Me0H (+10% NH4OH)
to
95% DCM, 5% Me0H (+10% NH4OH)). The fractions containing the product were
mixed and
concentrated to afford 2 fractions of intermediate 27 (680 mg, 50% yield, 96%
purity by LCMS
and 360 mg; 26% yield, 91% purity by LCMS)
Preparation of intermediate 28:
N R 0
NO N
I ,
,
N
A solution of intermediate 26 (2 g, 3.42 mmol) and TFA (2.9 mL, 37.9 mmol) in
DCM (29 mL)
was stirred at rt overnight. ACN was then added and the solution was
evaporated to dryness.
The residue was then dissolved in Et0Ac and iced water, basified with NH4OH.
The layers
were separated, and the aqueous layer was extracted with Et0Ac. The combined
organic layers
were dried over MgSO4, filtered and evaporated to give 1.80 g (98% yield) of
intermediate 28.
Preparation of intermediate 29:
NO N
N õN
Intermediate 27 (1.87 g, 3.20 mmol) in TFA (2.7 mL) and DCM (27 mL) was
stirred at rt
overnight. The solution was evaporated to dryness. The residue was then
dissolved in DCM and
iced water, basified with a 30% aqueous NH4OH solution. The aqueous layer was
extracted
with DCM The combined organic layers were dried over MgSO4, filtered and
evaporated to
give 1.35 g (78% yield) of intermediate 29 as a pale yellow solid.
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Alternative preparation of intermediate 29:
To a solution of intermediate 27 (1.40 g, 2.25 mmol) in acetone (30 mL) and
water (14 mL)
was added p-Toluenesulfonic acid (1.94 g, 11.276 mmol). The reaction solution
was stirred at
65 degrees for 5 hours. The resulting mixture was quenched with water and
ethyl acetate. The
combined organic layers were washed with water and brine, dried over anhydrous
sodium
sulfate. The solid was filtered off. The residue was concentrated under reduce
pressure to give
1.01 g (78%) of intermediate 29 as a yellow solid.
Preparation of intermediate 29a:
N RS
I
N,N)
Intermediate 29a was prepared accordingly to intermediate 28 starting from
intermediate 25
Example A9
Preparation of intermediate 30:
0¨
d_1_\AI RS
L)z
To a stirring solution of 2,6-diazaspiro[3.4]octane-6-carboxylic acid,
phenylmethyl ester (15 g,
60.9 mmol) in methanol (300 mL) was added intermediate 24 (13.61 g, 73.08
mmol) and acetic
acid (4.02 g, 66.99 mmol). After stirring for 0.5 hour at room temperature,
sodium
cyanoborohydride was added (7.65 g, 121.8 mmol). After stirring overnight at
50 C, the
reaction mixture was quenched with a potassium carbonate solution (10% in
water) and
extracted with ethyl acetate. The combined organic layers were washed with
brine and dried
over anhydrous sodium sulfate. The solid was filtered off. The filtrate was
concentrated under
reduced pressure. The residue was purified by silica gel column chromatography
(Mobile phase
A: PE; Mobile phase B: Et0Ac, eluent from 0-50% Et0Ac) to give 17.8 g (69%
yield) of
intermediate 30 as a light yellow oil.
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Preparation of intermediate 31:
s 0¨ N R
Cbz and intermediate 32: Cbz
170 g of intermediate 30 was purified by SFC with the following conditions:
Column:
CH1RALPAK_ 1G, 5 *25 cm,1 Oum; Mobile Phase A: CO2, Mobile Phase
B:Et0H:ACN:DCM=1:1:1; Flow rate:150 mL/min; Gradient:40% B; 220 nm; retention
time 1
= 4.45 min; retention time 2 = 5.88 min; Injection Volumn:3.8 ml; Number of
Runs:237 to give
two fractions. Fraction A: 67.0 g (>99% purity by LCMS, 39% yield, retention
time 2:5.88 min)
of intermediate 31 as a light-yellow oil. Fraction B: 65 g (99% purity, 38%
yield, retention time
1: 4.45 min) of intermediate 32 as a light-yellow oil.
Preparation of intermediate 33
so-
To a solution of intermediate 31(15 g, 36.01 mmol) in methanol (300 mL) was
added palladium
on activated carbon (10% palladium) (8g, 7.517 mmol). Then the mixture was
stirred at room
temperature for 5 hours under the hydrogen (2-3 atm.). The mixture was diluted
with methanol
and filtered through a pad of Celite . The filtrate was evaporated under
reduced pressure to
give 9.5 g of desired product as a yellow oil which was directly used in the
next step without
any further modifications.
Preparation of intermediate 34
S 0---
CI N
N,NCI
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To a solution of 3,5,6-trichloro-1,2,4-triazine (9.4 g, 50.99 mmol) in
dichloromethane (100 mL)
were added the mixture of intermediate 33 (12.0 g, 42.49 mmol) and
triethylamine (12 mL,
84.98 mmol) in dichloromethane (150 mL) under nitrogen at 0 C. After stirring
for 3 hours at
room temperature under nitrogen, the mixture was quenched with water and
extracted with
dichloromethane. The combined organic layers were dried over anhydrous sodium
sulfate. The
solid was filtered off The filtrate was concentrated under reduced pressure to
give 17.3 g (83%
yield, 88% purity by LCMS) of intermediate 34 as a yellow solid.
Preparation of intermediate 35.
S 0--
N
0 N
F Ni -1\1CI
A solution of intermediate 34 (1.6g; 3.72 mmol), intermediate 11 (1g; 4.44
mmol) and DBU
(2.7 mL; 18.45 mmol) in THE (150 mL) was stirred at rt for 72 hours. The
solution was poured
into cooled water and the product was extracted with Et0Ac. The organic layer
was dried over
MgSO4, filtered and evaporated to dryness. The crude (3g) was purified by
silica gel
chromatography (Stationary phase: irregular bare silica 80g, Mobile phase: 63%
Heptane, 2%
Me0H (+10% NH4OH), 35% Et0Ac). The fraction containing the product were mixed
and
concentrated to afford 1.48g (64% yield) of intermediate 35.
Alternative preparation of intermediate 35:
To a solution of intermediate 34(3.00 g, 6.971 mmol) and intermediate 11(1.88
g, 8.365 mmol)
in tetrahydrofuran (60 mL) was added tetramethylguanidine (1.37 g, 11.85
mmol). The reaction
solution was stirred for 2 days at room temperature. The resulting mixture was
quenched with
water and extracted with ethyl acetate. The combined organic layers were
washed with sodium
hydroxide (0.5 MIL), water and brine, dried over anhydrous sodium sulfate. The
solid was
filtered off The filtrate was concentrated under reduced pressure. The residue
was purified by
flash chromatography column (ethyl acetate/hexane 2:1) to give 2.60 g (59%
yield) of
intermediate 35 as a yellow solid.
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Preparation of intermediate 82:
N S
0
N,NCI
To a mixture of intermediate 34(10.0 g, 23.27 mmol) and intermediate 81 (5.89
g, 27.887 mmol)
in THE (250 mL) was added tetramethylguanidine (7.3 mL, 58.09 mmol). After
stirring at room
temperature for 48 hours, the reaction mixture was quenched with water and
extracted with
ethyl acetate. The combined organic layers were washed with water and brine
and dried over
Na2SO4, filtered and concentrated under reduced pressure. The residue was
purified by flash
silica gel column chromatography (Mobile phase A: PE; Mobile phase B: Et0Ac,
eluent from
0-93% Et0Ac) to give 7.5 g (49% yield) of intermediate 82 as a yellow solid.
Preparation of intermediate 83
S 0--
0
doN
NJ
To a mixture of intermediate 82 (7.0 g, 11.57 mmol) in tetrahydrofuran (140
mL) were added
1, l'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane
complex (472
mg, 0.58 mmol), sodium borohydride (744 mg, 19.67 mmol) and N,N,N',N'-
tetramethylethylenediamine (2.9 mL, 19.67 mmol). After stirring at room
temperature
overnight under the N2 atmosphere, the reaction mixture was quenched with
water and extracted
with ethyl acetate. The combined organic layers were washed with water and
brine and dried
over Na2SO4, filtered and concentrated under reduced pressure. The residue was
purified by
flash silica gel column chromatography (Me0H/DCM, 0%Me0H to 9%Me0H) to give
4.8 g
(54% yield, 85.1% purity based on LC/MS) of intermediate 83 as a brown solid.
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Preparation of intermediate 84:
N S
NO
N
I
To a mixture of intermediate 83 (4.8 g, 8.32 mmol) in acetone (100 mL) and
water (50 mL) was
added p-toluenesulfonic acid (7.17 g, 41.62 mmol). After stirring at 65 C
overnight, the
reaction mixture was quenched with saturated sodium bicarbonate solution and
extracted with
dichloromethane. The combined organic layers were washed with water and brine,
dried over
Na2SO4, filtered and concentrated under reduced pressure to give 3.1 g (45%
yield, 62.8%
purity based on LC/MS) of intermediate 84 as a brown solid.
Example Al 0
Preparation of intermediate 36:
\ /
0¨N
)/. _____________ CN¨Boc
0
To a stirring solution of 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid
(30.0 g, 149.09
mmol), 1-(3-dimethylaminopropy1)-3-ethylcarbodiimide hydrochloride (42.9 g,
223.64 mmol)
and N,0-dimethylhydroxylamine (21.8 g, 223.64 mmol) in DCM (500 mL) were added
NN-
diisopropylethylamine (61.7 mL,372.73 mmol) and 4-dimethylaminopyridine (3.6
g, 29.82
mmol). After stirring overnight at room temperature, the reaction solution was
diluted with
DCM (500 mL) and washed with water, 10% of citric acid aqueous solution, water
and brine,
dried over anhydrous sodium sulfate and filtered. The filtrate was
concentrated under reduced
pressure to give 24.0 g of intermediate 36 as a light-yellow oil.
Preparation of intermediate 37:
______________________ N¨Boc
0
To a stirred solution of intermediate 36 (26.5 g,108.5 mmol) in
tetrahydrofuran (250 mL) was
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added isopropylmagnesium chloride (271 mL, 542.0 mmol, 2M in TFIF) at 0 C.
After stirring
overnight at room temperature, the reaction mixture was quenched with brine
(300 mL) at 0 C
and extracted with ethyl acetate (3 x 500 mL). The combined organic layer was
washed with
water and brine, dried over anhydrous sodium sulfate and filtered. The
filtrate was concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography (PE/EA,
8:2) to give 19.5 g of intermediate 37 (87% purity, 68% yield) as a light
yellow oil.
Preparation of intermediate 39:
,Boc
Cbz
To a stirred mixture of tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate
(25.5 g, 120.12 mmol)
in tetrahydrofuran (250 mL) and potassium carbonate (36.52 g, 264.262 mmol) in
water (250
mL) was added benzyl chloroformate (20.3 mL, 144.143 mmol) at 0 C. After
stirring overnight
at room temperature, the reaction mixture was extracted with ethyl acetate (3
x 300 mL). The
combined organic layer was washed with water and brine, dried over anhydrous
sodium sulfate
and filtered. The filtrate was concentrated under reduced pressure. The
residue purified by silica
gel column chromatography (PE/EA, 6:4) to give 39.10 g of intermediate 39 (99%
purity, 93%
yield) as a light-yellow oil.
Preparation of intermediate 40:
NH
TFA salt
Cbz
To a solution of intermediate 39 (55.5 g, 160.2 mmol) in DCM (550 mL) was
added TFA (110
mL). After stirring for 2 hours at room temperature, the reaction solution was
concentrated. The
residue was dissolved in water (300 mL). The resulting aqueous solution was
basified to pH=8
with a saturated solution of NaHCO3 and extracted with DCM/Me0H (10:1, 4 x 500
mL). The
combined organic layer was washed with brine (2 x 300 mL), dried over
anhydrous Na2SO4
and filtered. The filtrate was concentrated under reduced pressure to give
45.3 g (74% yield) of
intermediate 40 as TFA salt and as a light brown solid.
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Preparation of intermediate 41:
N¨Boc
jN RS
Cbz
To a stirred solution of intermediate 40 (5.00 g, 13.88 mmol) in methanol (50
mL) was
intermediate 37 (3.79 g, 16.65 mmol). After stirring for 0.5 hour at room
temperature, sodium
cyanoborohydride (4.36 g, 69.38 mmol) was added. The resulting mixture was
stirring
overnight at 50 C. Additional intermediate 37 (1.58 g, 6.94 mmol) and sodium
cyanoborohydride (2.62 g, 41.63 mmol) were added. After stirring for 6 hours
at 50 C,
additional sodium cyanoborohydride (1.31 g, 20.814 mmol) was added. After
stirring overnight
at 50 C, the reaction mixture was quenched with saturated sodium bicarbonate
solution (100
mL) and extracted with ethyl acetate (3 x 300 mL). The combined organic layer
was washed
with water, brine and dried over anhydrous sodium sulfate and filtered. The
filtrate was
concentrated under reduced pressure. The residue was purified by silica gel
column
chromatography, eluted with (PE/EA, 7:3) to give 4.5 g (67% yield) of
intermediate 41 as a
light yellow oil
Preparation of intermediate 42:
S N¨Boc
N R
To a stirred solution of intermediate 41(3.60 g, 7.87 mmol) in ethanol (40 mL)
was added
palladium on activated carbon 10% Pd (800 mg). After stirring under a hydrogen
stream (2-3
atm) at room temperature for 2 hours, the reaction mixture was filtered
through a pad of Celite
which was washed with ethanol and DCM. The filtrate was concentrated under
reduced pressure
to give 2.5 g of intermediate 42 as a grey oil.
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Preparation of intermediate 43:
N¨Boc
N RS
CI, )=,,,
N
N CI
To a stirred solution of 3,4,6 -tri chl oropyri dazine (700 m g,2.
164 mmol) in N, N-
dim ethylformami de (15 ml) was added intermediate 42 (397 mg, 2.164 mmol) and
triethylamine (0.9 mL, 6.492 mmol). After stirring 3h at temperature, the
reaction mixture was
quenched with water (30 mL) and extracted with ethyl acetate (3 x 50 mL). The
combined
organic layer was washed with water, brine and dried over anhydrous sodium
sulfate and
filtered off The filtrate was concentrated under reduced pressure. The residue
was purified by
silica gel column chromatography, eluted with (PE: EA = 55:45) to give 900 mg
(84% yield)
of intermediate 43 as a white solid.
Preparation of intermediate 44:
N¨Boc
N RS
0
To a stirring solution of intermediate 43 (800 mg,1.701 mmol) in N,N-
dimethylacetamide (15
mL) were added intermediate 11(383 mg,1.70 mmol) and cesium carbonate (1.66 g,
5.10
mmol). After stirring for 3h at 130 C, the reaction mixture was cooled to room
temperature,
quenched with water (100 mL) and extracted with EA (3 x 80 mL).The combined
organic layer
was dried over anhydrous sodium sulfate and filtered. The filtrate was
concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography, eluted with
(PE: EA = 55:45) to give 800 mg (70% yield) of intermediate 44 as a white
solid.
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Preparation of intermediate 45:
N¨Boc
N RS
NO
o
To a stirred solution of intermediate 44 (750 mg, 1.138 mmol) in ethyl acetate
(15 mL) was
added palladium on activated carbon 10% Pd (800 mg). After stirring under a
hydrogen stream
(2-3 atm) at room temperature overnight, the reaction mixture was filtered
through a pad of
Celite which was washed with ethyl acetate and ethanol. The filtrate was
concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography, eluted with
(DCM: Me0H = 6:4) to give 303 mg of intermediate 45 (42% yield) as an off-
white solid.
Preparation of intermediate 50:
N¨Boc
N RS
0
N
N
A mixture of intermediate 18 (740 mg, 1.78 mmol), intermediate 37 (487 mg, 2.1
mmol),
NaBH3CN (337 mg, 5.4 mmol) and acetic acid (102 [iL, 1.78 mmol) in Me0H (15
mL) was
stirred at 50 C overnight. The reaction mixture was mixed with another
reaction performed on
220 mg of intermediate 18. The resulting reaction mixture was poured into ice
water, basified
with a saturated solution of NaHCO3 and DCM was added. The organic layer was
separated,
washed with brine, dried over MgSO4, filtered and evaporated till dryness. The
residue was
purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40m
24g MERCK,
Mobile phase: Gradient from 99% DCM, 1% MeON (+10% NH4OH) to 95% DCM, 5% MeON
(+10% NH4OH)). The fractions containing the product were mixed and
concentrated to afford
1.04g (93% yield) of intermediate 50.
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Preparation of intermediate 51:
N *R
NO
0
N
N
and intermediate 52:
N¨Boc
N *S
0
0
N
Intermediate 50 (1.04g) was purified by chiral SFC (Stationary phase:
CHIRALPAK IC 5i.tm
250*30mm, Mobile phase: 50% CO2, 50% Et0H (0.3% iPrNH2)). The fractions
containing the
products were mixed and concentrated to afford 411 mg (37% yield) of
intermediate 51 and
427 mg (38% yield) of intermediate 52.
Alternative preparation of intermediate 50:
Under N2 flow, intermediate 18(854 mg; 1.13 mmol) and intermediate 37 (385 mg;
1.7 mmol)
in TI-IF (15 mL) were stirred at rt for 24h. Then, sodium triacetoxyb orohydri
de (718 mg; 3.39
mmol) was added portionwise The mixture was stirred at room temperature for
24h. The
solution was poured out into cooled water, basified with a solution of NaOH 3N
and Et0Ac
was added. The organic layer was separated, dried over MgSO4, filtered and
evaporated to
dryness. The residue was purified by silica gel chromatography (Stationary
phase: irregular
SiOH 15-40[Im 12g, Mobile phase: Gradient from 99% DCM, 1% Me0H (+10% NH4OH)
to
95% DCM, 5% Me0H (+10% NH4OH)). The fractions containing the product were
mixed and
concentrated to afford 200mg (28% yield) of intermediate 50.
Example Al2
Preparation of intermediate 53
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CI
CI
N ,N<7--
Phosphorus oxychl ori de (9.42 g, 61.4 mmol) was added dropwi se to a 0 C
(ice/water) solution
consisting of 4-chloropyridazin-3-ol (2.00 g, 38.3 mmol) and ACN (20 mL).
Then, the reaction
mixture was heated and stirred at 80 C for 3 hours before cooling to RT. The
reaction mixture
was slowly poured into water (50 mL) and adjusted to pH=8 by the saturated
solution of sodium
bicarbonate. The mixture was extracted with DCM (50 mL x 3). The combined
organic layers
were dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give
the crude product which was purified by FCC (silica gel, Mobile Phase A: PE;
Mobile Phase
B: Et0Ac, eluent with 0-25% Et0Ac) to give the intermediate 53 (2.00 g, 88%
yield) as a
yellow solid.
Preparation of intermediate 54:
Boc
CIy
A stir bar, intermediate 53 (500 mg, 3.36 mmol), tert-butyl 2,6-
diazaspiro[3.4]octane-2-
carboxylate (712 mg, 3.35 mmol), triethylamine (1.02 g, 10.1 mmol) and dry DCM
(10 mL)
were added to a 40 mL glass bottle before the resultant mixture was stirred at
25 C for 8 h.
The mixture was diluted into DCM (20 mL) and washed with water (10 mL x 3).
The organic
layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to
give the crude which was purified by FCC (silica gel, Mobile Phase A: PE;
Mobile Phase B:
Et0Ac, eluent with 0-100% Et0Ac) to give the intermediate 54 (500 mg, 42%
yield) as a yellow
solid.
Preparation of intermediate 55
,Boc
0
0
N,
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A stir bar, intermediate 11(346 mg, 1.54 mmol), intermediate 54 (500 mg, 1.54
mmol), cesium
carbonate (1.51 g, 4.63 mmol) and dry N,N-dimethylformamide (10 mL) were added
to a 50
mL round-bottomed flask before the resultant mixture was heated and stirred at
130 C for 8 h.
The mixture was cooled to room temperature and concentrated under reduced
pressure to give
a residue. The residue was suspended into dichloromethane (20 mL) and washed
with water
(10 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered and
concentrated
under reduced pressure to give a crude which was purified by FCC (silica gel,
Mobile Phase A:
Et0Ac; Mobile Phase B: Me0H, cluent with 0-10% Me0H) to give the intermediate
55 (700
mg, 80% yield) as a yellow solid.
Preparation of intermediate 56:
NH
0
N,
F
A stir bar, intermediate 55 (700 mg, 1.36 mmol), trifluoroacetic acid (4 mL)
and dry
dichloromethane (2 mL) were added to a 25 mL round-bottomed flask before the
mixture was
stirred at 25 'V for 40 min. The mixture was concentrated under reduced
pressure to give a
residue. The residue was diluted into dichloromethane (20 mL) and pH
wasadjusted to pH =12
by a solution of sodium hydroxide (3 M, 8 mL). The aqueous layer was extracted
with
dichloromethane (10 mL x 2). The combined organic layers were dried over
anhydrous Na2SO4,
filtered and concentrated under reduced pressure to give intermediate 56 (600
mg, crude) as a
yellow oil.
Preparation of intermediate 57:
\ /
O¨N
N¨Boc
0 _________________
HATU (99.5 g, 262 mmol) was added in portions to a 0 C (ice/water) mixture
consisting of 1-
(tert-butoxycarbonyl)piperidine-4-carboxylic acid (50.0 g, 218 mmol), N,0-
dimethylhydroxylamine hydrochloride (23.4 g, 240 mmol), Et3N (90.9 mL, 654
mmol), and
dichloromethane (500 mL). The reaction mixture was stirred at room -
temperature for 12 hours.
The reaction mixture was concentrated to dryness under reduced pressure. The
residue was
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diluted with water (1500 mL) and extracted with dichloromethane (500 mL x 3).
The combined
organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated
to dryness under
reduced pressure to afford the crude product, which was purified by FCC
(silica gel, Mobile
Phase A: PE; Mobile Phase B: Et0Ac, eluent with 0-50% Et0Ac) to afford the
intermediate 57
(54 g, yield: 82%) as a yellow oil.
Preparation of intermediate 58:
cN-Boo
0
Intermediate 57 (54.0 g, 198 mmol) and THF (500 mL) were added into a 1 L
three-necked
round-bottomed flask. i-PrMgC1 (198 mL, 397 mmol, 2 M in THF) was added
dropwise into
the mixture at 0 C (ice/water) under N2. The mixture was stirred with warming
to room
temperature for 10 hours before pouring into water (2000 mL) and extracted
with Et0Ac (1000
mL x 3). The organic phase was washed with brine, dried over Na2SO4, filtered
and
concentrated under reduced pressure to give the crude which was purified by
flash column
chromatography on silica gel (silica gel, Mobile Phase A: PE; Mobile Phase B:
Et0Ac, eluent
with 0-35% Et0Ac) to give the intermediate 58 (19.2 g, 34% yield) as a yellow
oil.
Preparation of intermediate 59:
N-Boc
N RS
0
0
A stir bar, intermediate 58 (278 mg, 1.09 mmol), intermediate 56 (300 lug,
0.726 mmol), zinc
chloride (200 mg, 1.47 mmol) and dry methanol (6 mL) were added to a 40 mL
glass bottle
before the mixture was heated and stirred at 45 C for 4 h. Then, sodium
cyanotrihydroborate
(91.2 mg, 1.45 mmol) was added to the mixture. The resultant mixture was
stirred at 45 C for
another 40 h. The mixture was diluted into dichloromethane (40 mL) and washed
with water
(10 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered and
concentrated
under reduced pressure to give the crude which was purified by FCC (silica
gel, Mobile Phase
A: Et0Ac; Mobile Phase B: Me0H, eluent with 0-10% Me0H) to give intermediate
59 (150
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mg, 29% yield) as a yellow solid.
Example Al2
Preparation of intermediate 60:
N¨Boc
0
oy,N
I
Intermediate 18 (120 mg, 0.29 mmol), intermediate 58 (150 mg, 0.585 mmol) and
ZnC12 (80
mg, 0.59 mmol) were added to a 25 mL round bottomed flask and the resulting
mixture was
dissolved in Me0H (5 mL). The mixture was heated and stirred at 80 C for 4
hours. Sodium
cyanoborohydride (37 mg, 0.59 mmol) was added to the mixture. Then, the
mixture was stirred
at 80 C for 16 hours. Then, additional intermediate 58 (150 mg, 0.585 mmol),
ZnC12 (80 mg,
0.59 mmol), and NaBH3CN (37 mg, 0.59 mmol) were added into the above solution.
Then, the
mixture was stirred at 80 C for 6 hours. The reaction mixture was
concentrated to dryness
under reduced pressure to give the crude product which was purified by
preparative HPLC
using a Boston Green ODS 150 mm x 30 mm x 5 ttm column (eluent: 25% to 55%
(v/v) CH3CN
and H20 with 0.04%NH3H20+10mM NH4HCO3) to afford pure intermediate 60 which
was
suspended in water (10 mL). The mixture was frozen using dry ice/acetone, and
then lyophilized
to dryness to afford the intermediate 60 (60 mg) as a white solid.
Preparation of intermediate 60a
¨/
N'KIIII
N¨Boc
*R
N 0
I
N N
and intermediate 60b:
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N *S N¨Boc
N 0
01-"LN
I
Intermediate 60 (375 mg, 0.57 mmol) was purified by supercritical fluid
chromatography
(Separation condition: DAICEL CHIRALPAK IG (250 mm x 30 mm x 10 urn), Mobile
phase:
A: Supercritical CO2, B: 0.1%NH3H20 IPA, A:B =45:55 at 80 mL/min; Column Temp:
38 ;
Nozzle Pressure: 100Bar; Nozzle Temp: 60; Evaporator Temp: 20; Trimmer Temp:
25 ;
Wavelength: 220nm). The pure fractions were collected, and the volatiles were
removed under
vacuum. The resulting product was lyophilized to dryness to remove the solvent
residue
completely. Desired product intermediate 60a (15 mg, 4% yield) and
intermediate 60b (19 mg,
5% yield) were obtained as white solid.
Preparation of intermediate 61:
\ /
O¨N ifBoo
C
0
EDCI (34.0 g, 177 mmol) was added to a solution consisting of (R) - 1 -(tert-
butoxycarbonyl)pyrrolidine-3-carboxylic acid (25.0 g, 116 mmol), HOBT (24.0 g,
178 mmol),
DIPEA (102.5 mL, 586.9 mmol) and DMf (250 mL) at 0 C. The reaction mixture
was stirred
at for 5 min. /V, 0-dimethylhydroxylamine (12.5 g, 128 mmol) was added the
reaction mixture.
The reaction mixture was stirred at room-temperature for 10 h before cooling
to room
temperature. The mixture was poured into water (1000 mL) and extracted with
ethyl acetate
(400 mL x 3). The organic phase was washed with 5% aqueous citric acid
solution (400 mL x
3), sat. NaHCO3 (400 mL x 2), brine (400 mL x 2), dried over Na2SO4, filtered
and concentrated
under reduced pressure to give cnide intermediate 61 (26 g, 82% yield) as a
colourless oil.
Preparation of intermediate 62:
0
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i-PrMgC1 (101 mL, 202 mmol, 2 M, in THE) was added dropwi se to a 0 C
(ice/water) solution
of intermediate 61(26.0 g, 101 mmol) and THF (250 mL). The reaction mixture
was stirred at
room-temperature for 10 hours. The mixture was quenched with a saturated
solution of NH4C1
(500 mL) and extracted with ethyl acetate (500 mL x 3) .The combined organic
layers were
dried over anhydrous Na2SO4, filtered, and concentrated in vacuum to give the
product which
was purified by FCC (silica gel, Mobile Phase A: PE; Mobile Phase B: Et0Ac,
eluent with 0-
50% Et0Ac) to afford the intermediate 62 (15.0 g, 56% yield) as a yellow oil.
Preparation of intermediate 63:
,Boc
N
N
N
and intermediate 64:
Boc
N *S
0 4..N
I
To a solution of intermediate 18 (300 mg, 0.724 mmol) and intermediate 62 (524
mg, 2.17
mmol) in 15 mL of Me0H was added ZnC12 (395 mg, 2.90 mmol). After addition,
the reaction
mixture was stirred at 75 C for 3 hours, then NaBH3CN (182 mg, 2.90 mmol) was
added into
the reaction and the mixture was stirred at the same temperature for 4 hours.
Additional
intermediate 62 (300 mg) was added and the mixture was stirred at 75 C for 16
hours. The
reaction mixture was concentrated in vacuum and the residue was purified by
preparative HPLC
(Column Welch Xtimate C18 150 x 25mm x 5um, Mobile Phase A: water
(0.04%NH3H20+10mM NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 30
mL/min,
gradient condition from 61% B to 81% B). The pure fractions were collected,
and the solvent
was evaporated under vacuum. The aqueous layers were lyophilized to afford
intermediate 63
(75.0 mg, 16% yield) as a white solid and intermediate 64 (88 mg, 18% yield)
as a white solid.
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Preparation of intermediate 65:
\O-N/ -Boc
(3)/
(S)-1-(tert-Butoxycarbonyl)pyrrolidine-3-carboxylic acid (15.0 g, 69.7 mmol),
EDCI (20.039
g, 104.53 mmol), HOBT (14.125 g, 104.53 mmol) and DlEA (45.034 g, 348.44 mmol)
were
added to DIVif (100 mL) at 10 C. After 5 min N,0-dimethylhydroxylamine
hydrochloride
(7.477 g, 76.66 mmol) was added to the mixture. The mixture was stirred at 40
C for 10 h
before poured into water (400 mL) and extracted with ethyl acetate (300 mL x
3). The organic
phase was washed with 5% aqueous citric acid solution (3 x 300 mL), sat.
NaHCO3 (2 x 300
mL), brine (2 x 300 mL), dried over anhydrous Na2SO4, filtered and
concentrated under reduced
pressure to give the intermediate 65 (12.41 g, 74%) as a yellow oil.
Preparation of intermediate 66:
o/
The intermediate 65 (12.4 g, 48.0 mmol) and THF (20 mL) were added into a 250
mL round-
bottomed flask. Isopropylmagnesium chloride (49 mL, 98 mmol, 2 M in THE) was
added
dropwise into the mixture at 0 C (ice/water) under N2. The mixture was
stirred with warming
to room temperature for 10 h. The mixture was quenched with a saturated
aqueous solution of
NH4C1 (100 mL) and extracted with Et0Ac (200 mL x 3). The combined organic
extracts were
dried over anhydrous Na2SO4, filtered, and concentrated to dryness under
reduced pressure to
give the crude product which was purified by FCC (silica gel, Mobile Phase A:
PE; Mobile
Phase B: Et0Ac, eluent with 0-35% Et0Ac) to afford the intermediate 66 (7.6 g,
65%) as a
light yellow oil.
Preparation of intermediate 67:
Boc
/,.,
Nõ
0
I
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and intermediate 68:
*S
N 0
N
To a solution of intermediate 18 (300 mg, 0.724 mmol) and intermediate 66 (524
mg, 2.17
mmol) in 15 mL of Me0H was added ZnC12 (395 mg, 2.90 mmol). After addition,
the reaction
mixture was stirred at 75 C for 3 hours. Then, NaBH3CN (182 mg, 2.90 mmol)
was added into
the reaction mixture and the mixture was stirred at the same temperature for 4
hours. Additional
intermediate 67 (300 mg) was added and the mixture was stirred at 75 C for 16
hours. The
reaction mixture was cooled to 25 C and concentrated in vacuum. The residue
was purified by
preparative UPLC (Column Welch Xtimate C18 150 x 25mm x 5um, Mobile Phase A:
water
(0.04%NH3H20+10mM NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 30
mL/min,
gradient condition from 61% B to 81% B). The pure fractions were collected and
the solvent
was evaporated under vacuum. The aqueous layers were lyophilized to afford the
intermediate
67 (100 mg, 21% yield) as a white solid and the intermediate 68 (105 mg, 22%
yield) as a white
solid.
Preparation of intermediate 70:
N-13cic
N RS
N 0
ON
A mixture of 2-[(4-chl oro-5-pyrimi di nyl)oxy] -N-ethyl -5 -fluoro-N-(1 -m
ethyl ethyl)-b enzami de
(4.5 g, 13.322 mmol) and intermediate 42 (4.31 g, 13.322 mmol) in acetonitrile
(100 mL) was
added sodium carbonate (5.65 g, 53.29 mmol) at room temperature. After
stirring for 2 hours
at 90 C, the resulting mixture was cooled to room temperature and filtered
through a pad of
celite . The filtrate was concentrated under reduced pressure. The residue was
purified by silica
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gel column chromatography, eluted with (DCM/1V1e0H, 96.7:3.3) to afford 6.7g
(78% yield) of
intermediate 70.
Preparation of intermediate 71:
¨Boc
N *S
1
NO
N
)
and intermediate 72
________________________________ N¨Boc
*R
4,N)
)
Intermediate 70 (6.7g) was purified by via chiral SFC (Stationary phase:
CHIRACEL OJ-H
51.tm 250*30mm, Mobile phase: 94% CO2, 6% Me0H (0.3% iPrNH2)). The ft-action
containing
the product were mixed and concentrated to afford 3.18g (47% yield) of
intermediate 71 and
3.16g (47%yield) of intermediate 72.
Preparation of intermediate 73:
F
N¨Boc
0
EDCI (3.12 g, 13.7 mmol) was added to a solution of 1-(tert-butoxycarbony1)-3-
fluoroazetidine-3-carboxylic acid (2.00 g, 9.12 mmol), DIEA (6.5 mL, 36.7
mmol), N,0-
dimethylhydroxylamine hydrochloride (1.78 g, 18.2 mmol), and HOBT (1.85 g,
13.7 mmol) in
acetonitrile (20 mL) and the reaction mixture was allowed to stirred at 25 C
under N2 for 2 h.
The mixture was quenched with water (50 mL) and extracted with Et0Ac (100 mL x
3). The
Et0Ac layer was dried over Na2SO4, filtered and evaporated to give a residue,
which was
purified by FCC (silica gel, from PE:EA=100:0 to 60:40) to give intermediate
73 (1.5 g, 63%
yield) as a light yellow oil.
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Preparation of intermediate 74:
F \N¨Boc
0
Under N2 at 5 C, i-PrMgC1 2M in THF (10 mL, 20 mmol) was added to a solution
of
intermediate 73 (3.00 g, 11.4 mmol) in THF (30 mL) dropwise. The solution was
stirred at 5 C
for 30 min, allowed to slowly rise to 20 C and stirred for 12h. The reaction
mixture was poured
out into a mixture of ice water and a saturated aqueous NH4C1 solution and
extracted with
Et0Ac (200 mL x 2). The organic layer was decanted, dried over Na2SO4,
filtered and
evaporated to dryness. The resulting crude was purified by FCC (silica gel,
from PE: EA=100:0
to 70:30) to yield intermediate 74 (1.6 g, 51 % yield) as a colourless oil.
Preparation of intermediate 75:
N Boc
0
To a solution of bicyclo[1.1.1]pentane- 1 -carboxylic acid (1.00 g, 8.92
mmol), tert-butyl 4-
bromopiperidine- 1 -carboxylate (4.71 g, 17.8 mmol), 2,2'-bipyridine (696 mg,
4.46 mmol),
Ni(acac)2 (916 mg, 3.57 mmol), MgCl2 (2.55 g, 26.8 mmol), Zn (4.00 g, 61.2
mmol), 4A MS
(10.0 g) and DlEA (4.5 mL, 27.2 mmol) in THF/DMF (100 mL/30 mL) was added B
oc20 (7.79
g, 35.7 mmol) under an Ar atmosphere at 30 C. After addition, the reaction
mixture was stirred
at 30 C for 60 hours. The reaction mixture was poured into 150 mL of water
and extracted
with Et0Ac (150 mL x 2). The combined extracts were washed with brine (200
mL), dried over
Na2SO4, filtered and concentrated in vacuum. The resulting residue was
purified by column
chromatography (silica gel, eluent from PE/Et0Ac = 100:0 to 85:15) to afford
intermediate 75
(580 mg, purity 60% based on LCMS, 14% yield) as a colourless oil.
Preparation of intermediate 76:
NBoc
N RS
N 0
0 N
I
N
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To a solution of intermediate 75 (580 mg, 60% purity, 1.25 mmol), intermediate
18 (568 mg,
1.37 mmol) and AcOH (449 mg, 7.47 mmol) in 50 mL of Me0H was added NaBH3CN
(470
mg, 7.47 mmol). After addition, the reaction mixture was stirred at 60 C for
16 hours. The
reaction mixture was concentrated under vacuum and the residue was diluted
with 100 mL of
water and extracted with Et0Ac (100 mL x 2). The combined extracts were
concentrated in
vacuum and the resulting residue was purified by preparative HPLC (Column
Phenomenex
Gemini NX-C18 (75*30mm*3um), Mobile Phase A: water (0.2% FA), Mobile Phase B:
acetonitrile, Flow rate: 30 mL/min, gradient condition from 25%B to 55%B). The
pure fractions
were collected and lyophilized to afford intermediate 76 (310 mg, 37% yield)
as a white solid.
Preparation of intermediate 76:
Li¨CI
Mg-<>
Br
LiC1 (565.2 mg, 13.333 mmol) was dried under high vacuum by heating with a
heat gun and
allowed afterwards to cool to room temperature. Then, Mg turnings (324 mg,
13.333 mmol)
and THF (11.1 mL, 1 M, 11.1 mmol) were added. The reaction mixture was cooled
to 0 C and
bromocyclobutane (1.5 g, 11.1 mmol) was then added. The reaction mixture was
stirred at room
temperature for 2 hrs. In this time a grey solution was formed. The THF
solution of
cyclobutylmagnesium bromide. LiC1 or intermediate 76 (approx. 1 M) was used
directly in the
following reaction.
Preparation of intermediate 77:
NBoc
0
In flask, intermediate 57 (1.01 g, 3.704 mmol) was dissolved in dry THF (10
mL). The solution
was cooled in an ice bath and treated with a solution of freshly prepared
intermediate 76
(11.1mL, approx. 1 M, 11.1 mmol) dropwise at this temperature. The reaction
mixture was
stirred over night and was allowed to come to room temperature. Then saturated
ammonium
chloride solution was added, and the water phase was extracted with ethyl
acetate for three
times. After the organic phase was dried with magnesium sulfate and filtered,
the organic phase
was evaporated. The crude product (953 mg) was purified with flash CC (silica
gel, 15% EA in
n-heptane) to give 833 mg (28% yield) of intermediate 77 as a colorless oil.
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Preparation of intermediate 78:
NBoc
N RS
0
Oyk-,N
I
N,
intermediate 79:
[2:2
N'h-CNBoc
R'
0
0,
-1\1
N,N )
and intermediate 80:
NBoc
N s*
0
N
I
To a solution of intermediate 18 (90.0 mg, 0.217 mmol), 2 drops of acetic acid
and intermediate
77 (145.1 mg, 0.543 mmol) in methanol (4 mL) was added sodium cyanob orohydri
de (54.6 mg,
0.869 mmol). After stirring at 60 C overnight, the solvent was removed under
vacuum. Then,
the reaction was quenched with a saturated solution of sodium carbonate and
extracted with
ethyl acetate. The combined organic layers were washed with water, brine and
dried over
anhydrous magnesium sulfate. The solid was filtered off. The filtrate was
concentrated under
reduced pressure. The crude product (200 mg) was obtained as a colorless oil
and purified by
preparative CC (12 g silica gel, eluent from 2.5 to 5% Me0H in DCM) to give
intermediate 78
(106 mg, 73% yield) as a white solid. A enantiomeri c separation was performed
via Preparative
SFC (Stationary phase: Chiralpak Daicel IG 20 x 250 mm, Mobile phase: CO, Et0H
+ 0.4
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iPrNH2) yielding 224 mg of intermediate 79 and 261 mg intermediate 80
containing 5% of
intermediate 79.
Preparation of intermediate 85:
Boc
I X
To a mixture of 4-chloro-3-iodopyridine (2.00 g, 8.35 mmol) in DMF (30 mL) was
added tert-
butyl 2,6-diazaspiro[3.4]octane-2-carboxylate (1.95 g, 9.19 mmol) and Cs2CO3
(8.2 g, 25.2
mmol). The resultant mixture was stirred at 110 C overnight. The reaction was
cooled to room
temperature and diluted by water (100 mL), extracted with ethyl acetate (40 mL
x 3). The
combined organic layers were washed with brine (20 mL x 3), dried over
anhydrous Na2SO4,
filtered and concentrated under reduce pressure to give the crude which was
purified by FCC
(100% petroleum ether to petroleum ether: ethyl acetate = 1:1) to give
intermediate 85 (1.7 g,
purity 100%, yield 49%) as a white solid.
Preparation of intermediate 86:
,Boc
0
To a mixture of intermediate 11(2.72 g, 12.1 mmol) in N-methyl-2-pyrrolidone
(20 mL) was
added intermediate 85 (1.70 g, 4.09 mmol) and Cs2CO3 (4.00 g, 12.3 mmol). The
mixture was
replaced with argon. Then CuCl (255 mg, 2.58 mmol) and 2,2,6,6-tetramethy1-3,5-
heptanedione (0.4 mL, 1.91 mmol) was added under the protection of argon. The
resultant
mixture was stirred at 140 C overnight under argon atmosphere. The mixture
was cooled to
room temperature and diluted by water (100 mL), extracted with ethyl acetate
(40 mL x 3). The
combined organic layers was washed by brine (20 mL x 3), dried over anhydrous
Na2SO4,
filtered and concentrated under reduce pressure to give the crude, which was
purified by FCC
(100% DCM to DCM: Me0H = 10:1) to afford intermediate 86 (780 mg, purity
89.93%, yield
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33%) as a brown solid.
Preparation of intermediate 87:
0
To a mixture of intermediate 86 (200 mg, 0.390 mmol) in DCM (2 mL) was added
TFA (0.5
mL) at room temperature. The mixture was stirred at room temperature for 0.5
h. The reaction
mixture was evaporated under reduce pressure. The residue was diluted by 2 M
NaOH (5 mL),
extracted with DCM (10 mL x3). The combined organic layers was dried over by
anhydrous
Na2SO4, filtered and concentrated under reduce pressure to give intermediate
87 (160 mg, 99%
yield) as a yellow solid, which was used in next step without further
purification.
Preparation of intermediate 88:
Boc
N RS NH
N 0
To a mixture of intermediate 87 (160 mg, 0.388 mmol) in Me0H (4 mL) was added
intermediate 21(187 mg, 0.775 mmol) and AcOH (47 mg, 0.783 mmol). The mixture
was
stirred at 70 C for 1 h. Then the mixture was cooled to room temperature and
NaBH3CN (48
mg, 0.764 mmol) was added to the mixture. The resultant mixture was stirred at
70 C for
another 1 h. The reaction mixture was cooled to room temperature and
evaporated to remove
solvent. The residue was diluted by saturated NaHCO3 aqueous solution (10 mL),
extracted
with dichloromethane (10 mL x 3). The combined organic layers were washed with
brine (5
mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to give
the crude, which was purified by FCC (100% petroleum ether to 100% ethyl
acetate; TLC: ethyl
acetate, Rf = 0.1) to give intermediate 88 (100 mg, purity 99%, yield 40%) as
a yellow solid.
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Preparation of intermediate 89:
N¨Boc
N RS
0 J.,
N
F NN
To a solution of intermediate 19 (800 mg, 1.475 mmol) in Me0H (10 mL) was
added
intermediate 37 (838 mg, 3.686 mmol). The mixture was stirring at room
temperature for 1 h.
The mixture was added NaBHCN (556 mg, 8.848 mmol) at 0 C. The mixture was
stirring at
room temperature for overnight. The mixture was quenched with Sodium
bicarbonate solution,
extracted with EA, washed with water and brine, dried over Na2SO4, filtered
and evaporated
under reduced pressure. The residue was purified by flash chromatography
(silica gel, eluent
from 100% DCM to 10% Me0H in DCM) to give 300 mg of intermediate 89 as a
yellow oil.
B. Preparation of compounds
Preparation of compound 1:
RS
0
I
A mixture of 2-[(4-chl oro-5-pyrimi di nyl)oxy] -N-ethyl -5 -fluoro-N-(1 -m
ethyl ethyl)-b enzami de
(174 mg; 0.516 mmol), intermediate 4 (173 mg; 0.62 mmol) and sodium carbonate
(218 mg;
2.064 mmol) in ACN (20 mL) was refluxed (90 C) for 2 hours. The reaction
mixture was cooled
to RT, poured onto iced water and extracted with DCM. The organic layer was
decanted,
washed with water, filtered over Chromabond and evaporated to dryness. The
residue was
purified by chromatography over silica gel (irregular SiOH, 24g; mobile phase:
gradient from
0% NH4OH, 0% Me0H, 100% DCM to 1% NH4OH, 10% Me0H, 90% DCM). The pure
fractions were collected and evaporated to dryness yielding 150 mg of compound
1 (50% yield).
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Preparation of compound 2
0
N
LOLN
I
A mixture of 2-1(4-chl oro-5-pyri rni di nyl)oxy] -N-ethyl -5 -fluoro-N-(1 -m
ethyl ethyl)-b enzami de
(33.5 mg; 0.099 mmol), intermediate 8 (35 mg; 0.119 mmol) and sodium carbonate
(42 mg;
0.398 mmol) in ACN (3.7 mL) was refluxed (90 C) for 2h. A similar work up and
purification
than the one used to isolate compound 1 was applied and led to compound 2.
Preparation of compound 3:
0
*R
jr 21
'1\1
and compound 4:
ON
N-5-3
N *S
0
Compound 2 (361 mg) was purified via chiral SFC (Stationary phase: CHIRALPAK
AD-H
5pm 250*30mm, Mobile phase: 88% CO2, 12% Et0H (0.3% iPrNH2)). The fractions
containing the product were mixed and concentrated to afford 156 mg of a
fraction A which
was taken up with Et20 and evaporated till dryness to give 150 mg of compound
3 and 156 mg
of fraction B which was taken up with Et20 and evaporated till dryness to give
compound 4.
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Alternative preparation of compound 4:
The reaction was performed twice on (1.3g; 2.48 mmol) of compound 76.
Under N2 flow, NaBH(OAc)3 (1.56 g, 7.43 mmol) was added dropwise to a solution
of
compound 76 (2.6 g; 5 mmol), oxetane-3-carbaldehyde (0.37 mL; 5.35 mmol) in
THF (100
mL). Then, the reaction mixture was stirred at RT for 2h. Both reactions
(performed on 1.3g of
compound 76) were mixed with another reaction performed on 700 mg of compound
76 and
the resulting mixture was poured into ice water, basified with an aqueous
solution of K2CO3
10% and Et0Ac was added. The organic layer was separated, washed with brine,
dried over
MgSO4, filtered and evaporated till dryness to give 3.93 g of crude compound 4
which was
mixed with additional 392 mg of crude compound 4. The resulting crude was
purified by silica
gel chromatography (Stationary phase: irregular SiOH 15-40 m 80g MERCK, Mobile
phase:
Gradient from 97% DCM, 3% Me0H (+10% NH4OH) to 90% DCM, 10% Me0H (+10%
NH4OH)). The fractions containing the product were mixed and concentrated to
afford 2.5 g of
compound 4 (white product) and 1.2 g of impure (71.6% purity evaluated by
LC/MC)
compound 4
Analogous reaction protocols as reported for compounds 1 and 2 could be used
for the
preparation of compounds listed in the table below starting from the
appropriate starting
materials.
C\.0
RS
N RSN
0 0
N
I
Compound 5 Compound 6
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\---0__\
7--- _/
'-,z_
\..--0_,
r
s CN N
si\N * --1 *R
7 7
N 0 4,N -õ,__N 0 N
0õA 0.,)-.
F I
Th\J F -` N
I
-1\1
Compound 7
Compound 8
-----/ H 1-1_
_ -
S S
N *R N *S
N N
N 0 --N,,,,-0
N N
I
FN Fõ---,,,õ--,.-- ----.N-;-
--
Compound 9
Compound 10
------H H
N------)R
*R N *S R
N N
--_,,N
N N
C:1)
0,AN N
1 I
F F 'N
Compound 11
Compound 12
/ 0 / CO
N I-1' R N I-1 R
N
N N
0.-1.,
F
-N-J 1
F N
Compound 13
Compound 14
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N
. -
Rh_ic2 *s
N H s N H s
--1 I
N 0 -...,..N 0
N N
F N-PI 1
F
Compound 15 Compound 16
o
0
N RS D N RS
D D
--,õ-
--,,,N 0 -,,,N 0
N N
F 1
F -" N
I
Compound 17 Compound 18
NS
--1 -'1
N
I N
I )
F N F N
Compound 19 Compound 20
CN
N *S
N
0,.),,,,
F I
Th\1
Compound 21
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Preparation of compound 22:
H2
N RS
N 0
0 N
I
NN
At 0 C, TFA (3.2mL; 42.2mmo1) was added to intermediate 22 (1.35g; 2.11mmol)
in DCM (32
mL). Then, the reaction was warmed to room temperature and stirred for 15h at
room
temperature.
The reaction mixture was concentrated, and the residue was dissolved in 40 mL
of water. The
solution was basified with a solution of NaOH 1M until pH=8-9. After stirring
for 10 min at
room temperature, the resulting mixture was extracted with DCM. The combined
organic layers
were washed with brine and dried over MgSO4, filtered and evaporated till
dryness to give
0.84g (74%) of compound 22.
Preparation of corn pound 32
NH
N RS
N 0
N,N
A solution of intermediate 45 (100 mg; 0.16 mmol) and TFA (0.25 mL; 3.27 mmol)
in DCM
(2.5 mL) was stirred at rt overnight. TFA was eliminated by evaporation. The
residue was taken
up with water, basified with an aqueous solution of NH4OH. The organic layer
was extracted
with DCM, dried over MgSO4 and evaporated to dryness to give 84 mg of compound
32
(quantitative).
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Preparation of compound 33:
NH
N RS
N 0
N
N N
At 0 C, TFA (0.49 mL; 6.4 mmol) was added to a solution of intermediate 50
(200 mg; 0.32
mmol) in DCM (7 mL). The reaction mixture was stirred overnight at RT. The
residue was
evaporated till dryness. The residue (420 mg) was purified by silica gel
chromatography
(Stationary phase: irregular SiOH 15-40m 12g, Mobile phase: Gradient from 90%
DCM, 10%
Me0H (+10% NH4OH) to 85% DCM, 15% Me0H (+10% NH4OH)). The fraction containing
the product were mixed and concentrated to afford 144 mg (85% yield) of
compound 33.
Preparation of compound 34:
H
N 0
0 N
N
NJ
At 0 C, TFA (0.42 mL; 5.5 mmol) was added to a solution of intermediate 51(173
mg; 0.28
mmol) in DCM (6 mL). The reaction mixture was stirred overnight at RT. The
solvent was
evaporated. The residue was dissolved in water. Then, the solution was
basified with a solution
of NaOH 1M until pH=9-10. After stirring for 10 min at room temperature, the
resulting mixture
was extracted with dichloromethane (3x). The combined organic layer was washed
with brine
and dried over MgSO4, filtered and evaporated till dryness to give 150 mg
(quantitative) of
compound 34.
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Preparation of compound 35:
r\T7c;NH
N 0
N
N
NJ
Compound 35 was prepared accordingly to compound 34 starting from intermediate
52.
Preparation of compound 36:
NH
N RS
TEA salt
N 0
0
N
A stir bar, intermediate 59 (130 mg, 0.199 mmol), trifluoroacetic acid (2 mL)
and dry
di chl oromethane (1 mL) were added to a 25 mL round-bottomed flask before the
resultant
mixture was stirred at 25 C for 1 h. The mixture was concentrated under
reduced pressure to
give e compound 36 (130.0 mg, crude) as colourless oil which was used to the
next step directly
without further purification.
Preparation of compound 37:
NH
_Li¨N RS
N 0
0 N
N
Ni
Intermediate 60 (40 mg, 0.061 mmol), 1,4-dioxane (0.5 mL) and HC1/1,4-dioxane
(0.2 mL, 4
M) were added to a 10 mL round-bottomed flask. The reaction mixture was
stirred at room-
temperature for 12 hours. The reaction mixture was concentrated to dryness
under reduced
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pressure to afford the title compound which was dissolved in H20 (10 mL). The
resultant
solution was basified with solid NaHCO3 to pH = 8 and extracted with ethyl
acetate (10 mL x
3). The combined organic extracts were dried over anhydrous Na2SO4, filtered,
and
concentrated to dryness under reduced pressure. The residue was suspended in
water (10 mL).
The mixture was frozen using dry ice/acetone and then lyophilized to afford
compound 37 (30
mg, crude), as a white solid which was used in the next step without further
purification.
Preparation of compound 78:
N17:FTCNH
HCI salt
NO
F NN
HC1/dioxane (200 uL, 0.400 mmol, 2M) was added to a solution consisting of
intermediate 60a
(15 mg, 0.023 mmol) in dioxane (1 mL). The reaction mixture was stirred at
r.t. for 2 hr. White
solid was precipitated. The solvent was removed by syringe and the white solid
was
concentrated to dryness under reduced pressure to afford the title compound
which was
suspended in water (10 mL) and frozen using dry ice/ethanol, and then
lyophilized to dryness
to afford the compound 78 (6.47 mg, 47% yield) as a white solid.
Preparation of compound 79:
NH
N *s
HCI salt
NO N
N,
HC1/dioxane (200 uL, 0.400 mmol) was added to a solution consisting of
intermediate 60b (19
mg, 0.029 mmol) and dioxane (1 mL). The reaction mixture was stirred at r.t.
for 2 h. White
solid was precipitated. The solvent was removed by syringe and the white solid
was
concentrated to dryness under reduced pressure to afford the title compound
which was
suspended in water (10 mL) and frozen using dry ice/ethanol, and then
lyophilized to dryness
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to afford compound 79 (7.32 mg, 42% yield) as a white solid.
Preparation of compound 45:
¨/
CNN
*R
1
4.N HCl salt
NO
N,N!)
HC1/dioxane (150 uL, 0.300 mmol, 2 M) was added to a solution consisting of
intermediate 63
(20 mg, 0.031 mmol) and dioxane (1 mL). The reaction mixture was stirred at
room-temperature
for 4 hours. The white solid was precipitated. The solvent (dioxane) was
removed by syringe
and the white solid was concentrated to dryness under reduced pressure to
afford the title
compound which was suspended in water (10 mL). The mixture was frozen using
dry
ice/ethanol and then lyophilized to dryness to afford the compound 45 (3.17
mg, 17% yield) as
a white solid.
Preparation of compound 46
_______________________________ FC_IJVH
N *S
HCI salt
F NN
0
HC1/dioxane (150 uL, 0.300 mmol, 2 M) was added to a solution consisting of
intermediate 64
(19 mg, 0.030 mmol) and dioxane (1 mL). The reaction mixture was stirred at
room-temperature
for 4 hours. The white solid was precipitated. The solvent (dioxane) was
removed by syringe
and the white solid was concentrated to dryness under reduced pressure to
afford the title
compound which was suspended in water (10 mL). The mixture frozen using dry
ice/ethanol
and then lyophilized to dryness to afford the compound 46 (8.01 mg, 46% yield)
as a white
solid.
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Preparation of compound 49:
/
N *R
ii
HCl/1,4-dioxane (0.3 mL) was added to the mixture consisting of intermediate
67 (25 mg, 0.039
mmol) and 1,4-dioxane (1 mL) at 0 C. The resultant mixture was stirred at
room-temperature
for 1 h. The reaction mixture was concentrated to dryness under reduced
pressure to afford the
crude product which was purified by preparative HPLC using a YMC-Triart Prep
C18 250 x
50 mm x 10 .ina column (eluent: 45% to 75% (v/v) CH3CN with 0.04% NH3H20-E 1
OmM
NH4HCO3) to afford pure product. The product was suspended in water (10 mL).
The mixture
was frozen using dry ice/ethanol, and then lyophilized to dryness to afford
the compound 49
(3.88 mg, 89%. purity based on LC/MS, 16% yield) as a white solid.
Alternative procedure for the preparation of compound 49.
A stir bar, intermediate 67 (70.0 mg, 0.109 mmol) and hydrochloric
acid/dioxane (2 mL, 8.0
mmol, 4 M in dioxane) were added to a 10 mL round-bottomed flask before the
mixture was
stirred at 25 C for 1 h. The mixture was concentrated under reduced pressure
to give 70 mg of
crude compound 49 (HC1 salt) as a white solid which was used in the next step
without further
purification.
Preparation of compound 50:
NH
N *S
F
0
N,
To a solution of intermediate 68 (40.0 mg, 0.063 mmol) in 1 mL of dioxane was
added
HC1/dioxane (3 mL). After addition, the reaction mixture was stirred at 10 C
for 45 minutes.
The reaction mixture was concentrated in vacuum and the residue was purified
by preparative
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HPLC (Column Boston Prime C18 150 x 30mm x 5um, Mobile Phase A: water
(0.04%N1-13H20+10mM NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 25
mL/min,
gradient condition from 45% B to 75%). The pure fractions were collected, and
the solvent was
evaporated under vacuum. The aqueous layer was lyophilized to afford compound
50 (12 mg,
34% yield) as white solid.
Preparation of compound 76:
N H
N 0
0
In a round bottom flask, at 0 C, TFA (12.7mL; 166 mmol) was added to
intermediate 71(5.18
g; 8.29 mmol) in DCM (175 mL). Then, the reaction was warmed to room
temperature and the
reaction mixture was stirred overnight at room temperature. The residue was
dissolved in 20
mL of water. Then, the solution was basified with a solution of NaOH 1M (70
mL) until pH=8-
9. After stirring for 10 min at room temperature the resulting mixture was
extracted with
dichloromethane (5 x 100 mL).The combined organic layers were washed with
brine (1 x 150
mL) and dried over MgSO4, filtered and evaporated till dryness to give 4 g
(92% yield) of
compound 76.
The compounds listed in the table below were prepared following analogous
reaction
protocols as reported for the preparation of compounds 76 starting from the
corresponding
starting materials.
Compound number Compound structure
¨/
NI/
77 NO N
N
From intermediate 72
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¨/
F
/ \L \
¨NR NH
88 0
01),N
I
NN
From intermediate 18 and intermediate 74
F\L
N S NH
89 N 0
0,T)N
I
NN
From intermediate 18 and intermediate 74
Preparation of compound 80:
NH
N RS
TFA salt
N 0
F NN
O'rLN
To a solution of intermediate 76 (380 mg, 0.561 mmol) in 3 mL of DCM was added
TFA (6
mL). After addition, the reaction mixture was stirred at 27 C for 1 hour.
The reaction mixture
was concentrated in vacuum to afford compound 80 (350 mg, crude, TFA salt)
which was used
in next step without any purification.
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Preparation of compound 112:
NH
N *S
0
0 N
1
N
NJ
In a vial intermediate 80 (208 mg, 0.312 mmol) was dissolved in DCM (3.00 mL,
46.9 mmol)
and cooled to 0 C. The mixture was treated with TFA (0.478 mL, 6.25 mmol),
then the cooling
bath was removed. After stirring overnight sat. sodium carbonate solution was
added as well as
DCM. The water phase was further basified with 1 N aqueous NaOH solution to pH
13. The
water phase was extracted multiple times with DCM and then with ethyl acetate.
The collected
organic solvents were dried with MgSO4, filtered, then the solvents were
removed to yield
compound 112 (150 mg, 85% yield) as a white solid.
Preparation of compound 113:
N *R
0
0 N
1
In a vial intermediate 79 (224 mg, 0.336 mmol) was dissolved in DCM (3.23 mL,
50.5 mmol)
and cooled to 0 C. The mixture was treated with TFA (0.515 mL, 6.73 mmol).
The cooling
bath was removed. After stirring overnight saturated sodium carbonate solution
was added as
well as DCM. The water phase was further basified with 1 N aqueous NaOH
solution to pH 13.
The water phase was extracted multiple times with DCM and ethyl acetate. The
collected
organic solvents were dried with MgSO4, filtered, then the solvents were
removed to yield
crude compound 113 (239 mg). 35 mg of crude compound 113 were used and
purified via
preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10nm,30x150mm,
Mobile
phase: 0.25% NH4HCO3 solution in water, CH3CN) yielding 16 mg of compound 113
as white
solid.
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Preparation of compound 54:
N 0
0
N.N
compound 55:
)1rii oI
N S NO-d's
N 0
oIN
NNJ
and compound 56
HH
oI
N S
0
(DN
I
N
Ni
In a sealed tube, NaBH3CN (45.8 mg, 0.729 mmol) was added to a mixture of
intermediate 29
(157 mg, 0.291 mmol), (S)-3-methoxypyrrolidine (88.4 mg, 0.874 mmol) and
acetic acid (16.7
p,L, 0.291 mmol) in methanol (4 mL). The reaction mixture was stirred at 60 C
for 18 h. A
saturated aqueous solution of NaHCO3 and Et0Ac were added. The layers were
separated. The
aqueous layer was extracted with Et0Ac. The combined organic layers were
washed with brine,
dried over MgSO4, filtered, concentrated and purified by silica gel
chromatography (irregular
SiOH 40 Jim, 12 g, liquid loading (DCM), mobile phase gradient: from
DCM/(Me0H/ aq. NH3:
9/1): 99/1 to 90/10). The fractions containing product were evaporated to give
164 mg of
compound 54 which was purified by reverse phase (Stationary phase: YMC-actus
Triart C18
10 i_tm 30*150 mm, Mobile phase: Gradient: (aq. NH4HCO3 0.2%,
pH=9.5)/(MeCN/MeOH:
1/1): from 40/60 to 10/90). The fractions containing products were evaporated,
solubilized in
MeCN, extended with water and freeze-dried to give 81 mg (45% yield) of
compound 55 as a
white fluffy solid and 22 mg (12% yield) of compound 56 as a white fluffy
solid.
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Preparation of compound 57:
H cis
N S No
N 0
N
I
N
NJ
and compound 58:
¨001 H cis
N S 0
N 0
I
NN
NaBH3CN (47 mg; 0.75 mmol) was added to a mixture of intermediate 29 (200 mg;
0.37 mmol),
(cis)-hexahydro-1H-furo[3,4-c]pyrrole (0.13 mL; 1.12 mmol) and AcOH (21 pL;
0.37 mmol)
in THU (10 mL) and the reaction mixture was stirred at 60 C for 18 h. The
reaction mixture
was cooled to RT, poured onto a 10% aqueous solution of K2CO3 and Et0Ac. The
organic layer
was decanted, separated, dried over MgSO4, filtered and evaporated to dryness
g. The crude
(340 mg) was purified by silica gel chromatography (Stationary phase:
irregular bare silica 12g,
Mobile phase: Gradient from 99% DCM, 1% Me0H (+10% NH4OH) to 90% DCM, 10%
Me0H (+10% NH4OH)). The fractions containing the product were mixed and
concentrated to
afford an intermediate fraction (220mg) which was purified via reverse phase
(Stationary phase:
YMC-actus Trion C18 15 p.m 35*220 mm, Mobile phase: Gradient from 40% (aq.
NH4HCO3
0.2% pH=9.5)/MeCN/Me0H to 40/30/30 to 20/40/40). yielding 135 mg of compound
57 which
were freeze dried with acetonitrile/water 20/80 to give 120 mg (55 % yield) of
compound 57 as
a white powder and 40 mg of compound 58 which were freeze-dried with
acetonitrile/water
20/80 to give 38 mg (16% yield) of compound 58 as a white powder.
The compounds listed in the table below were prepared following the same
procedures as
reported for the preparation of compounds 54, 55 and 56 starting from the
corresponding
starting materials.
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Compound number Compound structure
0
N S
59 0
0 N
I
NN
From intermediate 29 and 4-(isopropylsulfonyl)piperidine
H H
N
60 0
NN
From intermediate 28 and 4-(isopropylsulfonyl)piperidine
0-
-00Fri
N S
61 N 0
N
I
NN
From intermediate 29 and 2-methoxy-N-methylethan-1-amine
0
N S N N
62 NO
Cj'srj'N
I
N
F- N-
From intermediate 29 and 1-(piperazin-1-yl)ethan-1-one
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Compound number Compound
structure
H H H =
cis
63 0
N
I
N,N
H H H =
cis
NN 0
N 0
64
0 N
I
N,N=-;)
From intermediate 28 and
(c, i s)-h exahy dro-1H-furo [3 ,4-c]pyrrol e
FjoitiN
N S 0
N 0
HH
0 N
N,N
N S 0
N 0
0 N
66 F
I
N
From intermediate 29 and
2-oxa-6-azaspiro[3.31heptane
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Compound number Compound structure
H H
N R 0
67 N 0
0)A N
I
NN
H H
NNKO
0
68 o N
I
From intermediate 28 and
2-oxa-6-azaspiro[3.3]heptane
N S
69
N 0
(:)))N
F NN
N
N S H
0
0=.y.k.,
70 N
I
N,N-;)
From intermediate 29 and
cyclopropanamine
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Compound number Compound structure
HH
S.
RS N 0
N 0
74
I
N,N
HH
N 0
N RS
75 0
0 N
I
N,N_4)
From intermediate 29a and
morpholine
0
N S
127
0
I
0
N S
\T/
0
128
F NN
From intermediate 12, intermediate 34 and
1-(2,6-diazaspiro[3.31hept-2-yeethenone, TFA salt
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Compound number Compound structure
N S
N 0
129 0)AN
I
N,N
/
N S 0
130
0
0)AN
I
From intermediate 29 and 4-methoxypiperidine
Preparation of compound 131:
Ncis ,0
N S
/1\1-
-,N 0
N
I
and compound 132:
,0
HN
N S N
0
Oyt. N
I
To a solution of intermediate 29 (120 mg, 0.22 mmol) in methanol (2 ml) was
added the cis-
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N,N-dimethy1-3-azabicyclo[3.1.0]hexane-6-carboxamide (41 mg, 0.27 mmol). After
stirring for
20 minutes at room temperature, sodium cyanoborohydride (28 mg, 0.47 mmol) was
added to
the mixture. After stirring at 50 C overnight, the resulting mixture was
quenched with a
saturated sodium bicarbonate solution and extracted with dichloromethane. The
combined
organic layers were washed with water and brine and dried over anhydrous
sodium sulfate. The
solid was filtered off The filtrate was concentrated under reduced pressure
and the resulting
residue was purified by preparative HPLC with the following conditions
(Column: XSelect
CSH Prep C18 OBD Column, 5um,19*150mm ; Mobile Phase A: Water(lOmmon NH4HCO3),
Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 30%B to 60%B in 7 min;
220 nnr,
retention time 1: 6.35 min; retention time 2: 6.90 min. Mixed of the pure
fractions and
lyophilization afforded 46.5 mg (30.6% yield, retention time 1: 6.35 min) of
compound 131 as
white solid and 3 mg (1.9% yield, retention time 2: 6.90 min) of compound 132
as white solid.
Preparation of compound 120:
H cis
N S 0
0
N,
and compound 121:
H cis
N S 0
F NN-7
To a solution of intermediate 84 (2.5 g, 2.99 mmol, 62.8% purity) in Me0H (50
mL) was added
cis-hexahydro-1H-furo[3,4-c]pyrrole hydrochloride (1.07 g, 7.15 mmol). After
stirring at room
temperature for 30 minutes, NaBH3CN (599 mg, 9.53 mmol) was added to the
reaction mixture.
The resulting reaction mixture was stirred at 50 'V overnight and quenched
with saturated
sodium bicarbonate solution and extracted with dichloromethane. The combined
organic layers
were washed with water and brine, dried over Na2SO4, filtered and concentrated
under reduced
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pressure to give 800 mg of crude product as a yellow solid. The crude product
was purified by
preparative-HPLC ( YMC-Actus Triart C18, 30 mm X 150 mm, 5um; Mobile Phase
A:Water(10 mmol/L NH4HCO3+0.1%NH3.H20), Mobile Phase B:ACN; Flow rate:60
mL/min;
Gradient:40%B to 60%B in 7 min; 254 nm; RT1: 6.95 min; RT2: 8.27 min). The
fractions
containing the products were mixed. The solvents were concentrated and both
compounds were
freeze dried to afford to give 102.6 mg of compound 120 as a white solid and
23.1 mg of
compound 121 as a white solid.
CONVERSION
Preparation of compound 23:
F-joFt_i
N H
N RS
N 0
0 N
I
F" N"
compound 24:
0
H H c_)
N H
N 0
0
NI
N,N
and compound 25:
¨00;1 0
N *S
N H
N 0
0 N
N,N
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At room temperature, NaBH(OAc)3 (144 mg; 0.68 mmol) was added to a solution of
compound
22 (240 mg; 0.445 mmol) and tetrahydro-4H-pyran-4-one (48 !IL; 0.53 mmol) in
dichloroethane (6 mL). The mixture was stirred at rt overnight. The solution
was cooled and
poured into cooled water, basified with K2CO3 powder and the product was
extracted with
DCM. The organic layer was dried over MgSO4, filtered and evaporated to
dryness giving 380
mg of compound 23
Separation of the enantiomers (380 mg of compound 23) was performed via chiral
SFC
(Stationary phase: Chiralpak IG 5 m 250*20mm, Mobile phase: 50% CO2, 50% Et0H
(0.3%
iPrNH2)). The fractions containing the products were mixed, concentrated and
freeze-dried with
a mixture of acetonitrile/water (20/80) to afford 90 mf (32% yield) of
compound 24 as a white
powder and 98 mg (35% yield) of compound 25 as a white powder.
The compounds listed below were prepared by using analogous reaction protocols
as reported
for compound 23 starting from the respective starting materials
Compound
Structure
number
/
NH RS
N RS
Compound
N 0
26
Oyk,,
I IN
N
From compound 22 and dihydro-3(2H)-furanone
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N1-1 *s
N RS
Compound
0-1-1)k-N
27
N
0
/
NH *R
N RS
0 ,N)
Compound
28
From chiral SFC separation of compound 26: Stationary phase:
Chiralpak IG 5pm 250*30mm, Mobile phase: 55% CO2, 45%
mixture of Et0H/DCM 80/20 v/v +0.3%iPrNH2
0
H H
NH * R
NM? Or¨
Compound
N 0
29
N
I
NH *R
*S
Compound 0
30 N
I
N
NJ
From chiral SFC separation of compound 28: Stationary phase:
CH1RALPAK AD-H 5nm 250*21.2mm, Mobile phase: 85% CO2,
15% Et0H 0.3% iPrNH2
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NH
N RS
Compound N 0
31 oI N
N N
From compound 22 and oxetane-3 -carbaldehyde using NaBH3CN
as reductive agent
Preparation of compound 38:
0
N RS
M
NO
0
N N
Acetic acid (16 L; 0.28 mmol) was added at room temperature to a solution of
compound 32
(88 mg; 0.17 mmol) and oxetane-3-carbaldehyde (24 L; 0.35 mmol) in THF (3
mL). The
mixture was stirred at rt for 4h. Then, NaBH(OAc)3 (107 mg; 0.51 mmol) was
added
portionwise. The mixture was stirred at room temperature for 2 hours and then,
poured into ice
water, basified with a 10% aqueous solution of K2CO3. Et0Ac was added. The
organic layer
was separated, washed with brine, dried over MgSO4, filtered and evaporated
till dryness. The
resulting residue (98 mg) was purified by silica gel chromatography
(Stationary phase: irregular
SiOH 40 um 4 g, Mobile phase: Gradient from 100% DCM to 80% DCM, 20% Me0H
(+10%
NH4OH)). The fractions containing the product were mixed and concentrated to
give 60 mg of
compound 38 which was further purified via reverse phase (Stationary phase:
YMC-actus Triart
C18 lOnm 30*150mm, Mobile phase: Gradient from 40% NH4HCO3 0.2%, 60% Me0H to
20% NH4HCO3 0.2%, 80% Me0H). The fractions containing the product were mixed
and
concentrated to give 26 mg of compound 38 which was freeze-dried with
acetonitrile/water
(20/80) to give 24 mg (25% yield) of compound 38 as a white powder.
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Preparation of compound 39:
N-1-3
N RS
N 0
N
N
NJ
Acetic acid (24 ttL; 0.42 mmol) was added at room temperature to a solution of
compound 33
(144 mg; 0.27 mmol) and oxetane-3-carbaldehyde (38 L; 0.55 mmol) in THE (4
mL). The
mixture was stirred at rt overnight then NaBH(OAc)3 (177 mg; 0.83 mmol) was
added
portionwise. The mixture was stirred at room temperature for 24 hours. The
mixture was poured
into iced water. The aqueous layer was basified with K2CO3 powder and the
mixture was
extracted with Et0Ac (x2). The organic layers were combined, dried over MgSO4
and
evaporated till dryness. The residue (137 mg) was purified by silica gel
chromatography
(Stationary phase: irregular SiOH 15-40[tm 12g, Mobile phase: Gradient from
97% DCM, 3%
Me0H (+10% NH4OH) to 90% DCM, 10% Me0H (+10% NH4OH)). The fractions containing
the product were mixed and concentrated to give 90 mg of an intermediate
impure fraction
which was further purified by reverse phase (Stationary phase: YMC-actus
Triart C18 10um
30*150mm, Mobile phase: Gradient from 65% NH4HC030.2% , 35% ACN to 25% NH4HCO3
0.2%, 75% ACN). The fraction containing the product were mixed and
concentrated and the
resulting residue (44 mg) was freeze-dried with Acetonitrile/water (20/80) to
give 42 mg (26%
yield) of compound 39.
Preparation of compound 40:
N *R
N 0
F
N
I
N
NaBH(OAc)3 (91 mg; 0.43 mmol) was added dropwise to a solution of compound 34
(150 mg;
0.28 mmol) and oxetane-3-carbaldehyde (214; 0.3 mmol) in THF (6 mL). The
reaction
mixture was stirred at RT for 1.5 It The reaction mixture was poured into ice
water, basified
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with a solution of K2CO3 10% and Et0Ac was added. The organic layer was
separated, washed
with brine, dried over MgSO4, filtered and evaporated till dryness.The residue
(143 mg) was
purified by silica gel chromatography (Stationary phase: irregular SiOH 15-40
m 12g, Mobile
phase: Gradient from 97% DCM, 3% Me0H (+10% NH4OH) to 85% DCM, 15% Me0H (+10%
NH4OH)). The fractions containing the product were mixed, concentrated and the
resulting
residue (54 mg) was freeze-dried with acetonitrile/water (20/80) to give 50 mg
(29% yield) of
compound 40.
Preparation of compound 41:
0
N *s
0
I
NaBH(OAc)3 (82 mg; 0.39 mmol) was added dropwi se to a solution of compound 35
(135 mg;
0.25 minol) and oxetane-3-carbal dehyde (190,, 0.27 rnmol) in TT-1F (5 mT,).
The reaction
mixture was stirred at RT for 1.5 h. The reaction mixture was poured into ice
water, basified
with a solution of K2CO3 10% and Et0Ac was added. The organic layer was
separated, washed
with brine, dried over MgSO4, filtered and evaporated till dryness. The
residue (130 mg) was
purified by silica gel chromatography (Stationary phase: irregular SiOH 15-
40pm 12g, Mobile
phase: Gradient from 95% DCM, 5% Me0H (+10% NH4OH) to 92% DCM, 8% Me0H (+10%
NH4OH)). The fractions containing the product were mixed and concentrated to
afford a
fraction of 84 mg which was taken up with Et20 and evaporated till dryness to
give 70 mg (45%
yield) of compound 41.
Preparation of compound 42:
0
d_r1 RS
áON
I
NO N
NJ
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A stir bar, compound 37 (130 mg, 0.195 mmol), oxetane-3-carbaldehyde (16.8 mg,
0.195
mmol), triethylamine (98.7 mg, 0.975 mmol) and dry dichloromethane (4 mL) were
added to a
8 mL glass bottle before the mixture was stirred at 25 'V for 1 h. Then,
sodium
cyanotrihydroborate (36.7 mg, 0.584 mmol) was added to the mixture. The
resultant mixture
was stirred at 25 C for another 1 h. The mixture was diluted into
dichloromethane (40 mL) and
washed with water (20 mL x 3). The organic layer was dried over anhydrous
Na2SO4, filtered
and concentrated under reduced pressure to give the crude which was purified
by preparative
HPLC (Column: Phenomenex Gemini-NX 150*30mm*5um, Mobile Phase A:
water(0.04%NH3H20+10mM w NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 30
mL/min, gradient condition from 43% B to 71% B). The pure fractions were
collected, and the
solvent was evaporated under vacuum to give a residue. The residue was
partitioned between
acetonitrile (2 mL) and water (10 mL). The solution was lyophilized to dryness
to give
compound 42 (24.34 mg, 19% yield) as a white powder.
Preparation of compound 43:
0
cif_\31 *R
0
N
N
and compound 44:
0
N-fr
N *S
0
N
F NN
NaBH(OAc)3 (120 mg, 0.566 mmol) was added in portions to a 0 C (ice/water)
solution
consisting of compound 37 (100 mg, crude), oxetane-3-carbaldehyde (30.0 mg,
0.348 mmol),
Et3N (100 uL, 0.719 mmol) and dichloromethane (5 mL). The resultant mixture
was stirred at
room-temperature of 1.5 hours. The reaction mixture was concentrated under
reduced pressure
to give the crude product, which was purified by preparative HPLC using a
Welch Xtimate C18
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150*25mm*5um (eluent: 38% to 68% (v/v) CH3CN and H20 with 0.04%NH3H20+10mM
NH4HCO3) to afford pure product which was suspended in water (10 mL). The
mixture frozen
using dry ice/acetone, and then lyophilized to dryness to afford a white solid
(80 mg) which
was further purified by SFC (DAICEL CHIRALPAK AD-H(250mm*30mm,5um), isocratic
elution: i-PrOH (containing 0.1% of 25% aq. NH3): supercritical CO2, 30%: 70%
to 30%: 70%
(v/v)). The pure fractions were collected, and the volatiles were removed
under reduced
pressure. The product was suspended in water (10 mL). The mixture was frozen
using dry
ice/acetone and then, lyophilized to dryness to afford compound 43 (37.00 mg,
41% yield) as a
white solid and compound 44 (33.96 mg, 38% yield) as alight yellow solid.
The compounds listed in the table below were prepared following analogous
reaction protocols
as reported for the preparation of compounds 39 or 42 starting from the
corresponding starting
materials.
Compound number Compound structure
0
N-5-3
D/ID
NO N
di RS
0
122 lfjL
From N-(ethyl-d5)-5-fluoro-2-hydroxy-N-
isopropylbenzamide, intermediate 13 and 1-
azeti di necarb oxyli c acid, 3 -(2-m ethyl -1-oxopropyl )-, 1,1-
dimethylethyl ester
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Compound number Compound structure
0
N RS
N 0
123 ON
N
From N-cyclopropy1-5-fluoro-2-hydroxy-7V-
isopropylbenzamide, intermediate 13 and 1-
Azetidinecarboxylic acid, 3-(2-methyl-1-oxopropy1)-, 1,1-
dimethylethyl ester
N RS
N 0
124
ON
I
NN
From intermediate 18 and 1-aetidinecarboxylic acid, 3-(2-
methyl-l-oxopropy1)-, 1,1-dimethylethyl ester using sodium
cyanoborodeuteride as reducing agent
0
N-fr
N RS
N 0
125
N
I
N
Ni
From intermediate 18 and intermediate 58 using sodium
cyanoborodeuteride as reducing agent
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Compound number Compound structure
0
DD RS
D
N 0
126
0 N
I
N
From N-(ethyl -d5)-5 -flu oro-2-hy droxy -N-
isopropylbenzamide, intermediate 13 and intermediate 58
Preparation of compound 47:
N
N
0
0 yk'= N
I
NN)
To a solution of compound 45 (70.0 mg, 0.130 mmol) and oxetane-3-carbaldehyde
(50 mg,
0.581 mmol) in DCM (5 mL) was added TEA (80.0 mg, 0.791 mmol). The mixture was
stirred
at rt for 10 mins, then NaBH3CN (100 mg, 1.59 mmol) was added. The reaction
mixture was
stirred at rt for 1 hour and then concentrated to give a residue. The residue
was purified by
preparative HPLC (Column: YMC-Triart Prep C18 250 x 50mm x 10um, Mobile Phase
A:
water (0.04%NH3H20+10mM NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 25
mL/min,
gradient condition: from 45% B to 75% B) to afford compound 47 (20.0 mg, 24%
yield) as
white solid.
Preparation of compound 48:
______________________________ 17Ã(7)
N *S
NO
I
N
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To a solution of compound 46 (75.0 mg, crude), oxetane-3-carbaldehyde (35.9
mg, 0.417 mmol)
and TEA (70.3 mg, 0.695 mmol) in 5 mL of DCM was added NaBH3CN. After
addition, the
reaction mixture was stirred at 10 C for 2 hours. The reaction mixture was
concentrated in
vacuum and the residue was purified by prep-HPLC (Column Boston Prime C18 150
x 30mm
x 5um, Mobile Phase A: water (0.04%NH3H20+10mM NH4HCO3, Mobile Phase B:
acetonitrile, Flow rate: 25 mL/min, gradient condition from 45% B to 75%). The
pure fractions
were collected and lyophilized to afford the compound 48 (7.0 mg) as white
powder.
Preparation of compound 51:
2 , No
N
0
N
I
N
A stir bar, the compound 49 hydrochloride (70.0 mg, 0.121 mmol), oxetane-3-
carbaldehyde
(15.7 mg, 0.182 mmol), sodium cyanoborohydride (15.3 mg, 0.243 mmol),
triethylamine (61.5
mg, 0.608 mmol) and dry dichloromethane (2 mL) were added to a 10 mL round-
bottomed
flask before the resultant mixture was stirred at 25 C for 1 h. The mixture
was concentrated
under reduced pressure to give the crude which was purified by preparative
HPLC (Column:
Boston Prime C18 150*30mm*5um, Mobile Phase A: water(0.04%NH3H20 10mM
NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 25 mL/min, gradient
condition from 40%
B to 70%). The pure fractions were collected, and the solvent was evaporated
under vacuum to
give a residue. The residue was partitioned between acetonitrile (2 mL) and
water (10 mL). The
solution was lyophilized to dryness to give the compound 51(14.3 mg, 19%
yield) as a white
powder.
Preparation of compound 52:
N *S
0 d---3
(Y-N
I
N
NJ
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To a solution of the compound 50 (50.0 mg, 0.093 mmol), oxetane-2-carbaldehyde
(23.9 mg,
0.278 mmol) and TEA (18.8 mg, 0.185 mmol) in 5 mL of DCM was added NaBH3CN
(29.1
mg, 0.463 mmol). After addition, the reaction mixture was stirred at 10 "V for
1 hour. The
reaction mixture was concentrated in vacuum and the residue was purified by
preparative HPLC
(Column Boston Prime C18 150 x 30mm x Sum, Mobile Phase A: water
(0.04%NH3H20+10mM NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 25
mL/min,
gradient condition from 45% B to 75% B). The pure fractions were collected and
lyophilized
to afford the compound 52 (8.38 mg, 1 15% yield) as white solid.
Preparation of compound 53:
0
N-fr
.cirl RS
NO
A stir bar, compound 36 (130 mg, 0.195 rnmol), oxetane-3-carbaldehyde (16.8
mg, 0.195
mmol), triethylamine (98.7 mg, 0.975 mmol) and dry dichloromethane (4 mL) were
added to a
8 mL glass bottle and the mixture was stirred at 25 'V for 1 h. Then, sodium
cyanoborohydride
(36.7 mg, 0.584 mmol) was added to the mixture which was stirred at 25 C for
another 1 h.
The mixture was diluted into dichloromethane (40 mL) and washed with water (20
mL x 3).
The organic layer was dried over anhydrous Na2SO4, filtered and concentrated
under reduced
pressure to give the crude which was purified by preparative HPLC (Column:
Phenomenex
Gemini-NX 150*30mm*5um, Mobile Phase A: water(0.04%NH3H2O-F10mM NH4HCO3),
Mobile Phase B: acetonitrile, Flow rate: 30 mL/min, gradient condition from
43% B to 71%).
The pure fractions were collected, and the solvent was evaporated under vacuum
to give a
residue which was partitioned between acetonitrile (2 mL) and water (10 mL).
The solution was
lyophilized to dryness to give compound 53 (24.3 mg, 19. yield) as a white
powder.
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Preparation of compound 71:
N *S
N
1\1,
Sodium triacetoxyborohydrate (50 mg; 0.24 mmol) was added at room temperature
to a solution
of compound 35 (60 mg; 0.11 mmol) and dihydro-3(211)-furanone (18 [IL; 0.23
mmol) in
dichloroethane (8 mL). The mixture was stirred at rt for 2.5 h. The solution
was poured into
cooled water, basified with K2CO3 powder and the product was extracted with
DCM. The
organic layer was dried over MgSO4, filtered and evaporated to dryness. The
crude (79 mg)
was purified via Reverse phase (Stationary phase: YMC-actus Triart C18 10 m
30*150mm,
Mobile phase: Gradient from 65% NH4HCO3 0.2%, 35% ACN to 35% NH4HCO3 0.2%, 65%
ACN). The fractions containing the product were mixed and concentrated to
afford 48 mg of
an intermediate fraction which was freeze-dried with acetonitrile/water
(20/80) to give 40 mg
(59 % yield) of compound 71 as a white powder and a mixture of two
diastereoisomers.
The compounds listed in the table below were prepared accordingly to compound
71 started
from the corresponding intermediates.
Compound number Structure
N *S
0
Compound 72 0
N,
From compound 35 and dihydro-2H-pyran-3(4H)-one
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Compound number Structure
RS
Compound 73 0 ) 0
I
N,N
From compound 35 and 1-(tetrahydro-2H-pyran-4-yl)ethanone
Preparation of compound 81:
NRS
0
1
N,FN
To a solution of compound 80 (350 mg, TFA salt, 0.506 mmol), oxetane-2-
carbaldehyde (200
mg, 2.32 mmol) and TEA (500 mg, 4.94 mmol) in 50 mL of DCM was added NaBH3CN
(200
mg, 3.18 mmol). After addition, the reaction mixture was stirred at 28 C for 3
hours. The
reaction mixture was filtered and the filtrate was concentrated in vacuum and
the residue was
purified by preparative-HPLC (Column Boston Prime C18 150*30mm*5um, Mobile
Phase A:
water (0.04%Nn31420+10mM Nn4HCO3), Mobile Phase B: acetonitrile, Flow rate: 25
mLimin,
gradient condition from 50%B to 80%B). The pure fractions were collected and
lyophilized to
afford compound 81(170 mg, 45% yield) as white solid.
Preparation of compound 82:
N *R
0
I
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and compound 83:
N *S
0
N
I
N
170 mg of compound 81 were separated by Supercritical Fluid Chromatography
(separation
condition: DA10EL CH1RALPAK AS-H (250mm*30mm, Sum; Mobile phase: A:
Supercritical
CO2, B: 0.1%NH3H20-ETOH, A:B =55:45 at 80 mL/min) to afford impure compound 82
(60
mg, 91.5% purity based on LCMS) and impure compound 83 (60 mg, 94.5% purity
based on
LCMS) both as white solid. The compound 82 (60 mg, 91.5% purity based on LCMS)
was
further purified by preparative HPLC (Column Boston Prime C18 150*30mm*5um,
Mobile
Phase A: water (0.04%N1-13H20+10mM NH4HCO3, Mobile Phase B: acetonitrile, Flow
rate: 30
mL/min, gradient condition from 50%B to 80%B) to afford compound 82 (40.0 mg,
27% yield)
as white solid_ The compound 83 (60 mg, 94.5% purity based on LCMS) was
further purified
by preparative }PLC (Column Boston Prime C18 150*30mm*5um, Mobile Phase A:
water
(0.04%Nfl3f120+10mM NFLIFIC03, Mobile Phase B: acetonitrile, Flow rate: 30
mL/min,
gradient condition from 50%B to 80%B) to afford compound 83 as white solid.
The compounds listed in the table below were prepared following analogous
reaction protocols
as reported for the preparation of compounds 81, 82 and 83 starting from the
corresponding
starting materials. Someone skilled in the art, will realize that in some
cases, additional
deprotection steps might be required to get the final compounds.
Compound number Compound structure
YoH
*R
84 NO
0 N
I
NN
From compound 78
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Compound number Compound structure
N¨YOH
N *S
85 0
N
N,N
From compound 79
S,
N*R
86 0
N
From compound 78
SIp
N *S
87 0
N
N_N
From compound 79
N RS
90 0
N
N
NJ
From compound 37
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Compound number Compound structure
<-(3>
91 N
- OH
N RS
N 0
N
I
N
From compound 37
F
Ni*R
92 N
N
I
N
From compound 88
N RS
¨N
H
93 0
0
r\I
I
N
NJ
From compound 37
N R
N Rs
94 NO N
I
F N
From compound 37
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Compound number Compound structure
N RS N
N/ )
N
1 F 1
N,
N-;,-=,
From compound 37
N
N RS
----9
-- N
96
N
o'IAN
1
F NN
From compound 37
F
____/----OH
N
N *S
.'1
N
1 1
F NN
From compound 89
NyNH
cill RS
-')
98 -õ ,N, 0
T N
1 1
F N,N-;)
From compound 37
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Compound number Compound structure
0
¨/
*R
99 N
O N
I
N,N
From compound 78
0
N *S
100 0
ON
N,N
From compound 79
Cr0
N H
N RS
101 0
O N
N,N
From compound 37
0
)NH
N
N RS
102 N
.0,
", IT N
F N
From compound 37
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Compound number Compound structure
¨/ HO OH
N *R
103 NO
)-A-'N
I
From compound 78
HO OH
N *S
104 NO
I
From compound 79
OH
N RS
105
0
ON
FU NN)
From compound 37
N RS
0
106 0
0
I
N,N
From compound 37
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Compound number Compound structure
ciNj1 RS N
HN-s-
-C)
107 N 0
N
N.N
From compound 37
N RS
N}-11
108 N 0,N
I N
I _1
F N
From compound 37
N RS 0
109 -õ_,N 0
N
From compound 37
0
N-5-3
d_11 RS
110
0
N
From compound 37
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Compound number Compound structure
N *R
F
N 0 0
1 1 1
--y-"t"-N
I
N,
From intermediate 13, intermediate 58 and 5-fluoro-
2-hydroxy-N-isopropyl-N-(2,2,2-
trifluoroethyl)b enzami de
Preparation of compound 114:
N *R
N
NI _Nr,--)
In a flask compound 113 (59 mg, 0.104 mmol) was dissolved in methanol (1.27
mL, 31.3 mmol)
and treated with oxetane-3-carbaldehyde (35.9 mg, 0.417 rnmol), sodium
cyanoborohydride
(32.8 mg, 0.521 mmol) and 2 drops of AcOH. The mixture was stirred over night
at 60 C. The
solvent was evaporated and then, a saturated sodium carbonate solution was
added along with
DCM. Then, the water phase was further basified with 1 N aqueous NaOH solution
until pH
13. The water phase was extracted multiple times with DCM and ethyl acetate.
Drying with
magnesium sulfate, filtration and evaporation of solvents afforded the crude
material that was
purified. The purification was performed via preparative HPLC (Stationary
phase: RP XBridge
Prep C18 OBD-10 m,30x150mm, Mobile phase: 0.25% NH4HCO3 solution in water,
CH3CN)
yielding 42 mg (63% yield) of compound 114 as a white solid.
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Preparation of compound 115:
NON
¨
N *S
0
I
In a flask, compound 112 (50 mg, 0.0884 mmol) was dissolved in Me0H (1.07 mL,
26.5 mmol)
and treated with formaldehyde, 37% aqueous. solution (0.132 mL, 1.77 mmol), 2
drops of
HOAc, and, then with sodium cyanoborohydride (27.8 mg, 0.442 mmol). The
mixture was
heated over 2 h at 60 C. The solvent was evaporated. Then, a saturated.
sodium carbonate
solution was added along with DCM. The water phase was basified with 1 N
aqueous NaOH
solution to pH 13. The water phase was extracted multiple times with DCM and
ethyl acetate.
Drying with magnesium sulfate, filtration and evaporation of solvents afforded
crude product
(60 mg). A purification was performed via preparative HPLC (Stationary phase:
RP XBridge
Prep C18 OBD-10 m,30x150mm, Mobile phase: 0.25% NH4HCO3 solution in water,
CH3CN)
affording of compound 115 as a white solid.
The compounds listed below were prepared following an analogous reaction
protocol as
reported for the preparation of compounds 114 and 115
Compound Number Compound Structure
530
*S
1,N)
117 N 0
0-1/L.14
I
NN
From compound 112 and oxetane-3-carbaldehyde
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N N-
"R
118 N 0
N
I
N
NJ
From compound 113 and formaldehyde, 37% aqueous. solution
Preparation of compound 116:
/ _______________________________________ 0
NO N
0 N
II
F N
In a flask, compound 112 (45 mg, 0.0795 mmol) was dissolved in dry DMF (1.23
mL, 15.9
mmol) and treated with DIPEA (0.0411 mL, 0.239 mmol) and bromomethoxyethane
(12.2 mg,
0.0875 mmol). The reaction was stirred for 3 h at 80 C. A saturated sodium
carbonate solution
was added along with DCM. Then, the water phase was basified with 1 N aqueous
NaOH
solution to p14 13 The water phase was extracted multiple times with DCM and
ethyl acetate.
Drying with magnesium sulfate, filtration and evaporation of solvents afforded
the crude
product. A purification was performed via preparative HPLC (Stationary phase:
RP XBridge
Prep C18 OBD-10 m,30x150mm, Mobile phase: 0.25% NH4HCO3 solution in water,
CH3CN)
affording 19 mg (yield 38%) of compound 116 as a white solid.
Preparation of compound 119:
0
NH
N RS
N 0
0 N
I
N,N
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To a solution of compound 37 (200 mg, 0.36 mmol) in ACN (5 mL) were added 2-
bromo-N,2-
dimethylpropanamide (98 mg, 0.54 mmol) and K2CO3 (250 mg, 1.81 mmol). After
stirring at
70 C overnight, the reaction mixture was quenched with a saturated solution
sodium
bicarbonate and extracted with ethyl acetate. The combined organic layers were
washed with
water and brine and dried over Na2SO4, filtered and evaporated under reduced
pressure. The
residue was purified by preparative-TLC (Me0H/DCM, 1:10). The obtained crude
product (200
mg; white solid) was purified by preparative -HPLC (Column: XBridge Shield
RP18 OBD
Column, 19*250mm,10um; Mobile Phase A:Water (10 mmol/L NH4HCO3), Mobile Phase
B:ACN; Flow rate:25 mL/min; Gradient:55%B to 65%B in 7 min; 254/220 nm;
RT:5.93 min).
The fractions containing the product were mixed and concentrated to afford
40.4 mg (16% yield)
of compound 119 as a white solid.
Preparation of compound 133:
N RS
0
0
To a mixture of intermediate 88(100 mg, 0.157 mmol) in DCM (3 mL) was added
TFA (1 mL)
at room temperature. The mixture was stirred at room temperature for 0.5 h.
The reaction
mixture was evaporated under reduce pressure. The residue was diluted by 2M
NaOH (5 mL),
extracted with DCM (5 mL x 5). The combined organic layers was dried over
anhydrous
Na2SO4, filtered, and concentrated to dryness under reduce pressure to afford
compound 133
(84 mg, 99.6% yield) as a yellow solid.
Preparation of compound 134:
HH
N RS
0
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To a mixture of compound 133 (84 mg, 0.156 mmol) in Me0H (2 mL) was added
formaldehyde
(257 mg, 3.17 mmol, 37% in water) and acetic acid (20 mg, 0.333 mmol). The
mixture was
stirred at room temperature for 30 minutes. Then NaBH3CN (20 mg, 0.318 mmol)
was added
to the mixture and the resultant mixture was stirred at room temperature for 1
h. The reaction
mixture was evaporated to remove solvent. The residue was diluted by 2M NaOH
(5 mL),
extracted with DCM (10 mL x 3). The combined organic layers were dried over
anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give the crude,
which was purified
by preparative HPLC (Column: Welch Xtimate C18 150*30mm*5 m, Mobile Phase A:
water
(0.05%NH3H20+10mM NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 30
mL/min,
gradient condition from 55% B to 85%) to afford two fractions. The pure
desired fraction were
collected and the volatile solvent was removed by evaporation. The aqueous
residue was
lyophilized to afford compound 134 (40 mg, 99.52% purity, yield 45%) as white
powder. The
impure desired fraction were collected and the volatile solvent was removed by
evaporation.
The aqueous residue was lyophilized to afford compound 134 (12 mg, yield 14%,
¨95% purity
by NAIR) as white powder.
Preparation of compound 135:
¨/
-2-10L-F1N/
N *R
0
and compound 136
HH
N *S
NO N
foiL
F
Compound 134 (40 mg, 0.071 mmol) was purified by SFC (column: DAICEL CHIRALCEL
OD-H (250mm*30mm,5um), Mobile phase: A: Supercritical CO2, B: 0.1%NH3H20 IPA;
Isocratic: A:B = 75:25; Flow rate: 80 mL/min) to afford two fractions. The
pure fractions of
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first peak were collected and the volatile solvent was evaporated under
vacuum. The residue
was treated with H20 (3 mL) and CH3CN (1 mL). The mixture was lyophilized to
give
compound 135 (11 mg, 98.17% purity, yield 27%) as white powder. The pure
fractions of
second peak were collected and the volatile solvent was evaporated under
vacuum. The residue
was treated with H20 (3 mL) and CH3CN (1 mL). The mixture was lyophilized to
give
compound 136 (10 mg, 96.28 purity, yield 24%) as a white powder.
Preparation of compound 137:
N--
N *3 N
0
0
I
ON
To a solution of compound 76 (200 mg, crude) and 3-(dimethylamino)propanoic
acid
hydrochloride (49.0 mg, 0.32 mmol) in DCM (10 mL) was added HATU (121 mg, 0.32
mmol)
and DIEA (0.21 mL, 1.26 mmol). The mixture was stirred for 16 hours at rt. 20
mL DCM and
mL H20 was added to the mixture solution. The mixture was extracted with DCM
(30 mL
x 2), the combined extracts were washed with brine (30 mL) and dried over
Na2SO4, the mixture
15 was filtered and the filtrate was concentrated in vacuum. The residue
was purified by pre-HPLC
(Column: YlVIC-Triart Prep C18 250*50mm*10um, Mobile Phase A: water
(0.04%NH3H20+10mM NH4HCO3), Mobile Phase B: acetonitrile, Flow rate: 25
mL/min,
gradient condition from 45% B to 75%). The pure fractions were collected and
the solvent was
lyophilized to give the title compound compound 137 (20 mg, 96.7% purity, 12%
yield) as a
20 light yellow solid.
Preparation of compound 138:
NH
N RS
F NN
0 N
I
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To a solution of intermediate 89 (150 mg, 0.23 mmol) in dichloromethane (5.0
mL) were added
Trifluoroacetic acid (1.7 mL) at 0 C. The resulting mixture was stirred at
room temperature
for 3 hours. The resulting mixture was concentrated under reduced pressure to
give 150 mg of
compound 138 (97.5% purity, as trifluoroacetate) as a colorless oil.
Preparation of compound 139:
0
N RS
OH
0
N,N
To a mixture of compound 138 (150 mg, 0.241 mmol, purity 86.63%), glycolic
acid (22 mg,
0.289 mmol) and N,N-dii sopropylethylamine (0.12 mL, 0.722 mmol) in NN-
dimethylformamide (2 mL) was added HATU (110 mg, 0.289 mmol) in portions at 0
C and
stirred for 2 hours at room temperature. The reaction mixture was quenched by
the addition of
water (5 mL) and extracted with ethyl acetate (4 x 5 mL). The combined organic
layers were
washed with water (3 x 20 mL), brine (20 mL) and dried over anhydrous sodium
sulfate.
Filtration, concentration and the residue was pirified by reverse flash
chromatography with the
following conditions: Column: SunFire C18 OBD Prep Column, 19 mm X 250 mm;
Mobile
Phase A :Water(0.1%NH4HCO3), Mobile Phase B: ACN; Flow rate: 20 mL/min;
Gradient: 15%
B to 40% B in 11 min; 254/220 nm; Rt: 9.12 min to afford 46.9 mg of compound
139 as a white
solid.
LCMS (Liquid chromatography/Mass spectrometry)
General procedure
The High-Performance Liquid Chromatography (HPLC) measurement was performed
using a
LC pump, a diode-array (DAD) or a UV detector and a column as specified in the
respective
methods. If necessary, additional detectors were included (see table of
methods below).
Flow from the column was brought to the Mass Spectrometer (MS) which was
configured with
an atmospheric pressure ion source. It is within the knowledge of the skilled
person to set the
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tune parameters (e.g. scanning range, dwell time...) in order to obtain ions
allowing the
identification of the compound's nominal monoisotopic molecular weight (MW).
Data
acquisition was performed with appropriate software.
Compounds are described by their experimental retention times (Rt) and ions.
If not specified
differently in the table of data, the reported molecular ion corresponds to
the [MH-H1+
(protonated molecule) and/or [M-H]- (deprotonated molecule). In case the
compound was not
directly ionizable the type of adduct is specified (i.e. [M-FNH4]', [M-FLIC00]-
, etc...). For
molecules with multiple isotopic patterns (Br, Cl..), the reported value is
the one obtained for
the lowest isotope mass. All results were obtained with experimental
uncertainties that are
commonly associated with the method used.
Hereinafter, -SQD" means Single Quadrupole Detector, "RT" room temperature, -
BEH"
bridged ethyl siloxane/silica hybrid, "FISS" High Strength Silica, "DAD" Diode
Array Detector.
Table la: LCMS Method codes (Flow expressed in mL/min; column temperature (T)
in 'V;
Run time in minutes). "TFA" means trifluoroacetic acid
Flow
Method
Run
Instrument Column Mobile phase Gradient
code
time
Column T
1 Waters: Waters: BEH A: 95% 84.2% A for
0.343 6.2
Acquity C18 (1.7[1m, CH3COONH4 0.49min, to 10.5%
UPLC - 2.1x100mm) 7mM / 5% A in 2.18min, held
DAD and CH3CN, B: for
1.94m i n, back
Quattro CH3CN to 84.2% A in
MicroTM 0.73min, held
for
0.73min.
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Flow
Method
Run
Instrument Column Mobile phase Gradient
code
time
Column T
Agilent Waters A: water with First, 100 % A
was 0.8 10
XBridge Shield 0.05% hold for 1 minute.
RP18 NH3 .H2 0; Then a gradient
2.1*50 mm, 5 B: acetonitrile was applied to
UM 40 % A and 60 % 40
B in 4 minutes and
then to 5% A and
95 % B in 2.5
minutes. Finally
return to 100% A
in 2 minutes and
hold for 0.5
minute. Post Time
is 0.5minute.
3 Agilent Waters A:
water with 100%A was hold 0.8 10
Xbridge-C18 0.04 % TFA; for 1 minute, A
2.1*50 mm, 5 mobile phase, gradient from
urn B: acetonitrile 100% A to 40% A
with 0.02% is applied in 4
TFA minutes, and
40%A down to
15%A in 2.5
minutes. And then
return to 100%A in
2 minutes and hold
for 0.5 minutes.
The post time is
0.5min.
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Flow
Method
Run
Instrument Column Mobile phase Gradient
code
time
Column T
4 Agilent Waters mobilephaseA:
First, 90 % A was 0.8 10
Xbridge-C18 waterwith hold for 0.8
2.1*50 mm, 5 0.04%TFA; minute. Then a
UM mobilephaseB: gradient was
acetonitrilewith applied to 20 % A
0.02%TFA and 80% B in 3.7
minutes and hold
for 3 minutes. And
then return to 90%
A in 2 minutes and
hold for 0.5
minutes. The post
time is 0.5min.
5 Waters: 0.5 3.3
A: 95% From 85%A/15%Bto
Acquity
Waters BEH CH3COONH4 10% A in 2.1min, held
UPLC H-
C18 (1.7um, 7mM / 5%
for2min,backto85% 40
Class -
2.1x100mm) CH3CN, B: A/15%B in
0.8min,
DAD and
CH3CN held for 0.7min.
QDa
6 Shimadzu Poroshell ACN-
Water- 0.0 min 10 % B -> L2 3.00
LCMS- HPH-C18 6.5 mM 2.0 min 95 % B
2020 NH4HCO3 + -
>2.6 min 95% B
NH3H20 -> 2.75 min 10%
B->3.00 min
Controller
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Flow
Method Run
Instrument Column Mobile phase
Gradient
code
time
Column T
7 Shimadzu Ascentis ACN -Water-
0.0 min 5 % B -> 1.5 3.00
LCMS- Express C18 0.05% TFA 2.0 min 95% B ->
2020 2.7 min 95% B-
>
2.8 min 5%
B->3.00 min
Controller
8 Shimadzu
Kinetex EVO ACN-Water- 0.0 min 5 % B -> 1.2 3.00
LCMS- C18 0.03% 2.0 min 95 % B
2020 NH3H20 ->2.7
min 95% B
-> 2.75 min 5% B
-> 3.00 min
Controller
9 Shimadzu Poroshell ACN-Water-
0.0 min 5 % B -> 1.2 3.00
LCMS- HPH-C18 5mM 2.0 min 95% B
->
2020 NH4HCO3 2.7 min 95%
B->2.75 min 5% B
->3.00 min
Controller
10 Shimadzu Poroshell ACN-Water-
0.0 min 10 % B -> 1.2 2.85
LCMS- HPH-C18 5mM 2.0 min 95% B
->
2020 NH4HCO3 2.7
min 95% B->
2.75 min 10% B
->2.85 min
Controller
11 Shimadzu Ascentis ACN-Water-
0.0 min 5 % B -> 1.5 3.00
LCMS- Express C18 0.05% TFA 2.0 min 100% B ->
2020 2.7 min 100%
B->2.75 min 5%
->3.00 min
Controller
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Flow
Method Run
Instrument Column Mobile phase Gradient
code
time
Column T
12 Shimadzu Kinetex EVO ACN-Water- 0.0 min 10 % B -> 1.2
2.85
LCMS- C18 100A 5mM 2.0 min 95 % B
2020 NH4HCO3 ->2.7 min 95% B
-> 2.75 min 10% B
->2.85 min
Controller
13 Shimadzu Gemini NX- ACN-Water- 0.0 min 5 % B -> 1.5
3.00
LCMS- C18 5mM 1.0 min 95% B ->
2020 NH4HCO3 2.7 min 95% B->
2.75 min 5% B
->3.00 min
Controller
14 Shimadzu Poroshell ACN-Water- 0.0 min 10 % B -> 1.2
3.00
LCMS- HPH-C18 6.5 mM 2.0 min 95 % B
2020 NH4HCO3 + ->2.7 min 95% B
NH3H20 -> 2.75 min 10% B
-> Controller
15 Waters: Waters : BEH A: 10mM 0.8
2.00
Acquity C18 CH3COONH4 From 95% A to
upLce - (1.7um, in 95% H20 + 5% A in 1.3min,
held for 0.7 min
DAD and 2.1*50mm) 5% CH3CN
SQD B: CH3CN
16 Waters: Waters :BEH A: 10mM From 100% A to 0.6
3.50
Acquity (1.8um, NH4HCO3 5% A in 2.10min,
UPLC - 2.1*100mm) in 95% H2 0 + t o 0% Amn 1.4 min 55
DAD and 5% CH3CN
SQD2 B: Me0H
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Flow
Method
Run
Instrument Column Mobile phase Gradient
code
time
Column T
17 Waters: Waters :BEH A: 0.1%
From 100% A to 0.6 3.50
Acquity (1.8 um, NH4HCO3 5% A in 2.10min,
UPLC - 2.1*100mm) in 95% H20 + to 0% A in 1.4min 55
DAD and 5% CH3CN
SQD2 B: CH3CN
18 ACQUITY Aquity UPLC A: CH3OH From 5% A to 95% 0.7 2.0
BEH C18 A in 1.20 min, held
UPLC B: 10mM
1.7um for 0.2 mm, to 5%
System 2.1x5Omm NH4Ac in A in 0.20 min. 70
with SQD- Column90% H20 and
detector 10% CH3CN
Waters BEH A: From 95% A/5% B 0.5
19 Waters:
3.5
Acquity C18 (1.7Pm, CH3COONH4 to 5% A in lmin,
H-Class - 2.1x50mm)
7mM 95%/ held for 1.6min, back
DAD CH3CN5%, to 95% A/5%B in 40
and
B: CH3CN 0.2min, held for
SQD2
0.5min.
Table lb: LCMS and melting point data. Co. No. means compound number; Rt means
retention
time in min.
Co. No. Rt (min) [M-I-H]' [M+CH3C00]- LCMS Method
1 2.32 581.5 639.7 1
2 2.47 595.7 653.7 1
3 2.38 595.7 653.8 1
4 2.38 595.7 653.9 1
2.46 609.6 667.8 1
6 2.46 607.6 665.6 1
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Co. No. Rt (min) [M+F-1] [M+CH3COO] LCMS Method
7 2.61 607.6 665.8 1
8 2.52 607.6 665.8 1
9 2.59 623.6 681.7 1
2.61 623.6 681.7 1
11 2.62 623.6 681.7 1
12 2.59 623.6 681.8 1
13 2.46 609.6 667.6 1
14 2.50 609.6 667.8 1
2.50 609.6 667.8 1
16 2.46 609.6 667.6 1
17 5.109 623.4 2
18 4.952 612.5 2
19 2.055 609.5 4
5.195 609.4 2
21 2.956 595.4 3
22 2.10 540.5 598.6 1
24 2.18 624.6 682.8 1
2.18 624.6 682.7 1
27 2.21 610.6 668.5 1
29 2.23 610.6 668.7 1
2.22 610.6 668.7 1
31 2.19 610.6 668.7 1
34 2.91 526.2 3
2.93 526.4 3
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Co. No. Rt (min) [M+F-I] [M+CH3COO] LCMS Method
38 2.31 595.5 653.7 1
39 2.31 596.6 654.7 1
40 2.32 596.6 654.7 1
41 2.33 596.6 654.7 1
42 2.999 624.3 3
43 3.040 624.3 3
44 3.032 624.3 3
45 2.935 540.4 3
46 2.953 540.4 3
47 4.635 610.4 2
48 4.688 610.4 2
49 2.950 540.4 3
50 3.008 540.4 3
51 3.031 610.4 3
52 3.081 610.4 3
53 2.99 623.5 3
55 2.40 624.7 682.5 1
56 2.58 624.6 682.5 1
57 2.37 636.7 694.8 1
58 2.53 636.7 694.7 1
59 2.50 714.8 772.8 1
60 1.27 714.5 19
61 2.40 612.6 670.5 1
62 2.29 651.7 709.8 1
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Co. No. Rt (min) [M+F-1] [M+CH3COO] LCMS Method
63 2.33 636.7 694.7 1
64 2.53 636.7 694.7 1
65 2.26 622.7 680.7 1
66 2.38 622.7 680.8 1
67 2.14 622.5 5
68 2.28 622.4 5
69 2.46 580.6 638.6 1
70 2.52 580.6 638.6 1
71 2.43 596.6 654.7 1
2.44; 668.7
72 610.6 1
2.50
73 2.47 638.6 696.8 1
74 2.41 610.6 668.6 1
75 2.50 610.6 669.4 1
76 1.09 525.4 583.4 19
77 1.15 525.5 583.6 19
78 2.979 554.4 3
79 2.989 554.4 3
82 4.710 648.3 2
83 4.711 648.3 2
84 3.053 624.5 3
85 3.079 624.5 3
86 3.096 675.4 3
87 4.852 675.4 2
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Co. No. Rt (min) [M+F-1] [M+CH3COO] LCMS Method
88 2.976 544.4 3
89 2.959 544.4 3
90 3.028 598.5 3
91 3.006 640.5 3
92 3.053 588.4 3
93 3.000 662.4 3
94 3.035 651.5 3
95 3.197 635.4 3
96 3.093 635.4 3
97 3.058 588.4 3
98 2.208 665.5 4
99 3.101 665.5 3
100 3.094 665.5 3
101 4.632 651.5 2
102 4.736 666.5 2
103 4.756 628.4 2
104 4.699 628.5 2
105 3.119 652.5 3
106 3.094 665.5 3
107 3.136 701.5 3
108 4.871 635.4 2
109 3.153 686.4 3
110 3.092 672.3 3
111 3.241 678.3 3
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Co. No. Rt (min) [M+F-1] [M+CH3COO] LCMS Method
112 0.82 566.4 15
113 2.10 566.7 16
114 2.39 636.8 16
115 0.86 580.5 15
116 0.89 624.5 15
117 0.86 636.5 15
118 1.84 580.5 17
119 2.026 653.25 9
120 1.406 622.45 12
121 1.497 622.45 12
122 1.413 601.50 6
123 4.484 608.45 6
124 0.794 597.65 7
125 1.851 625.10 8
126 1.688 629.55 8
127 1.397 677.45 10
128 0.825 677.40 11
129 1.185 638.35 13
130 1.809 638.35 14
131 1.596 677.35 14
132 1.654 677.40 9
134 2.884 566.3 3
135 2.872 566.6 3
136 2.868 566.4 3
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Co. No. Rt (min) [M+F-I] [M+CH3COO] LCMS Method
137 3.042 624.5 3
139 1.090 598.4 18
SFC-Methods
General procedure for SFC methods
The SFC measurement was performed using an Analytical Supercritical fluid
chromatography
(SFC) system composed by a binary pump for delivering carbon dioxide (CO2) and
modifier,
an autosampler, a column oven, a diode array detector equipped with a high-
pressure flow cell
standing up to 400 bars. If configured with a Mass Spectrometer (MS) the flow
from the column
was brought to the (MS). It is within the knowledge of the skilled person to
set the tune
parameters (e.g. scanning range, dwell time...) in order to obtain ions
allowing the
identification of the compound's nominal monoisotopic molecular weight (MW).
Data
acquisition was performed with appropriate software.
Table 2a. Analytical SFC Methods (Flow expressed in mL/min; column temperature
(T) in C;
Run time in minutes, Backpressure (BPR), "DEA" means diethylamine.
Run
Method Flow
column mobile phase gradient
time
code
Col T BPR
AD, 3 [1..m, 3.5
10
CO2/Et0H/iPrNH2
1 4.6*100
90/10/0.3
(CHIRALPAK)) 35
103 bar
from 5% to
4 4
40% of B in 2
Chiralpak AD-3 min and hold
A: CO2 B:iso-propanol
2 50x4.6mm ID., 40% for 1.2
(0 05% DEA)
1500
3um min, then 5% 35
(PSI)
of B for 0.8
min
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from 5% to
ChiralPak AD-3 40%
of B in 2.5 7 bar
A: CO2 B:Ethanol (0.05%
3 150x4.6mm I.D. 5.5min , then
DEA)
3um 5% of B 40
100 bar
for 1.5 min
from 5% to 4 4
40% of B in 4
1500
Chiralcel OJ-3 min
and hold 35
A: CO2 B:ethanol (0.05%
(PSI)
4 100x4.6mm ID., 40% for 2.5
DEA)
3um min, then 5%
35 100 bar
of B for 1.5
min
ChiralPAK IC-3 3.5
6
CO2/Et0H/iPrNH2
100x4.6mm I.D.
45/55/0.3 35
103 bar
3um.
ChiralPAK IG-3 3.5
6
CO2/Et0H/iPrNH2
6 100x4.6mm ID.,
60/40/0.3 35
103 bar
3um.
ChiralPAK AD-3 3.5
6
CO2/Et0H/iPrNH2
7 100x4.6mm I.D.
85/15/0.3 35
103 bar
3um
AD, 3 !lin, 3.5
6
CO2/Et0H/iPrNH2
8 4.6*100
45/55/0.3
(CHIRALPAK)) 35
103 bar
Instrument:
from 5% to 2.5 10
Waters UPCC
40% of B in 5
with PDA
A:Supercritical CO2, min and hold
Detector
9 Mobile phase B: iso- 40% for 2.5
Column:
1500
propanol (0.05% DEA) min,
then 5% 35
Chiralpak AD-3
(PSI)
of B for
150x4.6mm ID.,
2.5 min
3um
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Instrument:
1.5
10
Shimadzu LC-
20AB with PDA A: Hexane(0.1%DEA) 90% B hold 10
detector column: B:Et0H(0.1%DEA) min 25
7.40
Chiralcel OD
MPa
150*4.6mm 3um
Instrument:
2.8
10
Waters UPCC
with PDA
A: CO2 B:ethanol (0.05% hold 15% for
11 Detector column :
DEA) 10 min
1500
Chiralcel OD-3 35
(PSI)
100 x4.6mm ID.,
3um
Table 2b. SFC data.
Isomer elution SFC
Co. No. RI (min) UV% Area
order Method
3 5.28 100 1 1
4 7.46 97.26 2 1
7 2.14 100 1 7
8 2.59 100 2 7
9 3.49 100 3 5
10 4.36 100 4 5
11 3.03 100 2 5
12 2.75 100 1 5
13 3.50 100 3 6
14 3.04 100 2 6
2.69 100 1 6
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Isomer elution SFC
Co. No. Rt (min) UV% Area
order Method
16 4.02 100 4 6
17 1.37/1.43 46.25/53.75 racemate 2
19 1.53/2.02 99.66/0.34 1 4
20 3.82/4.09 49.225/50.775 racemate 3
21 1.42/1.53 87.43/12.57 1 2
40 3.35 100 2 8
41 2.96 100 1 8
43 4.806 100 1 9
44 5.253 100 2 9
45 6.86 100 2 10
46 6.14 99.19 1 10
47 4.953 93.671 2 3
48 4.682 90.26 1 3
49 7.048 99.11 2 11
50 5.929 97.40 1 11
OPTICAL ROTATION (OR)
Optical Rotation is measured with a polarimeter 341 Perkin Elmer. The
polarized light is passed
through a sample with a path length of 1 decimeter and a sample concentration
of 0.2 to 0.4
gram per 100 milliliters. 2 to 4 mg of the product in vial are weight, then
dissolved with 1 to
1.2 ml of spectroscopy solvent (DMF for example). The cell is filled with the
solution and put
into the polarimeter at a temperature of 20 C. The OR is read with 0.004 of
precision.
Calculation of the concentration: weight in gram x 100/ volume in ml
[a] d20 : (read rotation x 100) / (1.000 dm x concentration).
d is sodium D line (589 nanometer).
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Table 3. OR data: wavelength: 589 nm (specicied if different); solvent: DMF
(specicied if
different); temperature: 20 C; 'conc' means concentration (g/100 mL); 'OR'
means optical
rotation.
Co. No. OR ( ) Conc.
3 -15.14 0.284
4 +8.3 0.265
7 -10.38 0.26
8 +12.6 0.262
24 +15.72 0.318
25 -16.59 0.416
27 + 14.22 0.228
29 - 18.04 0.316
30 - 16.67 0.728
40 -12.5 0.26
41 +13.67 0.256
55 +12.75 0.251
56 -4.43 0.271
62 + 10.4 0.25
NMR
Some NMR experiments were carried out using a Bruker Avance 500 spectrometer
equipped
with a Bruker 5mm BYWO probe head with z gradients and operating at 500 MT-Tz
for the proton
and 125 MI-Iz for carbon. Chemical shifts (d) are reported in parts per
million (ppm). J values
are expressed in Hz.
NMR experiments were carried out using a Bruker Avance III 400 spectrometer,
using internal
deuterium lock and equipped with reverse double-resonance (H, 13C, SET) probe
head with z
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gradients and operating at 400 MHz for the proton and 100MHz for carbon.
Chemical shifts (d)
are reported in parts per million (ppm). J values are expressed in Hz.
Some NMR experiments were carried out using a Bruker Avance III 400
spectrometer at
ambient temperature (298.6 K), using internal deuterium lock and equipped with
BBO 400MHz
Si 5 mm probe head with z gradients and operating at 400 MHz for the proton
and 1001VIHz
for carbon. Chemical shifts (d) are reported in parts per million (ppm). J
values are expressed
in Hz.
Some NIVER experiments were carried out using a Varian 400-MR spectrometer at
ambient
temperature (298.6 K), using internal deuterium lock and equipped with Varian
400 4NUC PFG
probe head with z gradients and operating at 400 MHz for the proton and 100MHz
for carbon.
Chemical shifts (d) are reported in parts per million (ppm). J values are
expressed in Hz.
Some NIVIR experiments were carried out using a Varian 400-VNIVIRS
spectrometer at ambient
temperature (298.6 K), using internal deuterium lock and equipped with Varian
400 ASW PFG
probe head with z gradients and operating at 400 MHz for the proton and 100MHz
for carbon.
Chemical shifts (d) are reported in parts per million (ppm). J values are
expressed in Hz.
Compound 4
Major rotamer (75%)
1H NMR (400 MHz, DMSO-d6) 6 ppm 8.30 (s, 1H), 7.69 - 7.81 (m, 1H), 7.15 - 7.38
(m, 2H),
6.88 - 7.02 (m, 1H), 4.55 (dd, J=7 .7 , 5.9 Hz, 2H), 4.20 (t, J=5.5 Hz, 2H),
3.73 - 3.84 (m, 1H),
3.37 - 3.70 (m, 5H), 3.12 - 3.27 (m, 3H), 3.05 (br d, J=6.2 Hz, 1H), 2.92 -
3.02 (m, 3H), 2.86
(dt, J=14.1, 7.1 Hz, 1H), 2.73 (br t, J=7.2 Hz, 1H), 2.60 (br dd, J=8.7, 6.4
Hz, 1H), 2.54 (br d,
J=7.6 Hz, 2H), 2.23 -2.36 (m, IH), 2.12 (br d, J=9.4 Hz, 1H), 1.95 (br t,
J=7.0 Hz, 2H), 1.45 -
1.59 (m, 1H), 1.21 (br dõ/=3.4 Hz, 2H), 0.98- 1.15 (m, 7H), 0.72 - 0.80 (m,
6H)
Minor rotamer (25%)
1H NMR (400 MHz, DMSO-d6) 6 ppm 8.28 (s, 1H), 7.69 - 7.81 (m, 1H), 7.15 - 7.38
(m, 2H),
6.88 - 7.02 (m, 1H), 4.55 (dd, J=7 .7 , 5.9 Hz, 2H), 4.41 (dt, .1=13.8, 6.8
Hz, 1H), 4.20 (t, .1=5.5
Hz, 21-1), 3.37 -3.70 (m, 51-1), 3.12 - 3.27 (m, 31-I), 3.05 (br d, J=6.2 Hz,
1H), 2.92 - 3.02 (m,
3H), 2.86 (dt, J=14.1, 7.1 Hz, 1H), 2.73 (br t, J=7.2 Hz, 1H), 2.60 (br dd,
J=8.7, 6.4 Hz, 1H),
2.54 (br d, J=7.6 Hz, 2H), 2.23 -2.36 (m, 1H), 2.12 (br d, J=9.4 Hz, 1H), 1.95
(br t, J=7.0 Hz,
2H), 1.45- 1.59 (m, 1H), 1.21 (br d, J=3.4 Hz, 2H), 0.98 - 1.15 (m, 7H), 0.72 -
0.80 (m, 6H)
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Compound 38
1-1-1N1VIR (500 MHz, DMSO-d6) 6 ppm 8.4 (d, J=5.7 Hz, 1H), 7.2 - 7.4 (m, 3H),
6.5 (br d, J=5.7
Hz, 1H), 4.6 (dd, J=7 .7 , 5.8 Hz, 2H), 4.2 (td, J=6.0, 2.2 Hz, 2H), 3.3 - 3.8
(m, 7H), 3.0 - 3.1 (m,
5H), 2.9 (dt, J=14.3, 6.9 Hz, 1H), 2.7 -2.8 (m, 1H), 2.5 - 2.6 (m, 3H), 2.3 -
2.4 (m, 1H), 2.2 (dd,
J=9.5, 1.9 Hz, 1H), 2.0 (br t, J=6.8 Hz, 2H), 1.5 - 1.6 (m, 1H), 1.0 (br d,
J=6.3 Hz, 4H), 0.9 -
1.0 (m, 4H), 0.8 (dd, J=12.9, 6.9 Hz, 6H), 0.6 (br s, 2H)
Compound 41
I-HM/1R (500 MHz, DMSO-d6) 6 ppm 8.48 (s, 1H), 7.22 - 7.52 (m, 3H), 4.56 (br
t, J=6.8 Hz,
2H), 4.17 - 4.30 (m, 2H), 3.87 - 4.13 (m, 2H), 3.49 - 3.72 (m, 3H), 3.30 -
3.41 (m, 1H), 2.97 -
3.20 (m, 6H), 2.88 (dt, J=14.3, 6.9 Hz, 1H), 2.77 (br d, J=1.3 Hz, 114), 2.55 -
2.69 (m, 2H), 2.30
-2.37 (m, 1H), 2.17 (br d, J=7.6 Hz, 1H), 1.96 - 2.10 (m, 2H), 1.51 - 1.66 (m,
1H), 0.92- 1.15
(m, 8H), 0.65 -0.84 (m, 9 H)
Compound 42
1H NMft (400 MHz, CDC13) 6 ppm 8.43 - 8.36 (m, 1H), 7.23 - 7.16 (m, 1H), 7.13 -
7.06 (m,
1H), 7.05 - 6.98 (m, 1H), 6.33 - 6.28 (m, 1H), 4.83 - 4.76 (m, 2H), 4.44 -
4.34 (m, 2H), 4.01 -
3.89 (m, 1H), 3.75 -3.56 (m, 4H), 3.55 - 3.47 (m, 1H), 3.27 - 3.05 (m, 6H),
2.82 - 2.72 (m, 2H),
2.66 (d, J = 7.2 Hz, 2H), 2.14 - 2.06 (m, 2H), 1.93 - 1.72 (m, 4H), 1.50 -0.98
(m, 12H), 0.93 -
0.84 (m, 6H), 0.71 (d, J= 6.4 Hz, 2H).
Compound 43
1-1-1NMIR (400 MHz, CDC13) 6 ppm 8.50 (s, 1H), 7.26 - 7.19 (m, 1H), 7.18 -7.09
(m, 1H), 7.08
- 6.97 (m, 1H), 4.89 - 4.73 (m, 2H), 4.49 - 4.34 (m, 2H), 4.33 - 4.01 (m,
2H), 4.00 - 3.83 (m,
1H), 3.81 -3.60 (m, 2H), 3.59 - 3.38 (m, 1H), 3.31 - 3.03 (m, 6H), 2.86 -2.61
(m, 4H), 2.21 -
2.07 (m, 2H), 1.96 - 1.71 (m, 5H), 1.56 - 1.33 (m, 5H), 1.15 - 1.05 (m, 6H),
0.93 - 0.84 (m, 6H),
0.82 - 0.72 (m, 2H)
Compound 44
1-H N1VIR (400 MHz, CDC13) 6 ppm 8.53 -8.44 (m, 1H), 7.27 - 7.18 (m, 1H), 7.18
-7.09 (m,
1H), 7.08 -6.97 (in, 111), 4.81 (t, J= 6.8 Hz, 2H), 4.51 -4.35 (in, 2H), 4.30 -
3.84 (m, 3H), 3.79
-3.55 (m, 2H), 3.54 - 3.41 (m, 1H), 3.38 - 3.06 (m, 6H), 3.01 -2.62 (m, 4H),
2.21 -2.11 (m,
2H), 2.11 - 1.64 (m, 5H), 1.64 - 1.29 (m, 5H), 1.14 - 1.03 (m, 6H), 0.95 -0.85
(m, 6H), 0.82 -
0.70 (m, 2H).
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Compound 45
IH NMR (400 MHz, CD30D) 6 ppm 8.87 - 8.69 (m, 1H), 7.67 - 7.42 (m, 1H), 7.39-
7.18(m,
2H), 4.66 -4.15 (m, 6H), 4.12 - 3.76 (m, 2H), 3.74 - 3.53 (m, 4H), 3.52 - 3.31
(m, 3H), 3.26 -
3.15 (m, 1H), 3.08 -2.94 (m, 1H), 2.79 -2.23 (m, 3H), 2.21 - 2.09 (m, 1H),
2.00 - 1.85 (m, 1H),
1.31 - 0.86 (m, 15H)
Compound 46
NMR (400 MHz, CD30D) 6 ppm 8.95 - 8.76 (m, 1H), 7.74 - 7.41 (m, 1H), 7.39 -
7.22 (m,
2H), 4.69 - 4.09 (m, 6H), 4.06 - 3.78 (m, 2H), 3.76- 3.50(m, 4H), 3.50 - 3.32
(m, 3H), 3.26 -
3.10 (m, 2H), 2.80 - 2.27 (m, 3H), 2.25 - 2.09 (m, 1H), 1.93 - 1.73 (m, 1H),
1.33 - 0.96 (m,
15H).
Compound 47
41 NMR_ (400 MHz, CD30D) 6 ppm 8.50 (s, 1H), 7.19-7.25 (m, 1H), 7.13 (s, 1H),
6.98-7.07
(m, 1H), 4.78 (t, J= 6.8 Hz, 2H), 4.37-4.45 (m, 2H), 3.95-4.25 (m, 2H), 3.82-
3.93 (m, 1H),
3.73 (s, 1H), 3.44 - 3.68 (m, 2H), 3.20 (d, J = 9.2 Hz, 6H), 2.62-2.82 (m,
4H), 2.27 (d, J= 6.4
Hz, 211), 2.06-2.19 (m, 3H), 2.03 (s, 1H), 1.89 (s, 1H), 1.67-1.81 (m, 2H),
1.01-1.22 (m, 7H),
0.72-0.91 (m, 8H).
Compound 48
NMR (400 1VIHz, CDC13) 6 ppm 8.50 (br. s, 1H), 7.18-7.25 (m, 1H), 7.08-7.17
(m, 1H),
6.94-7.07 (m, 1H), 4.69-4.87 (m, 2H), 4.37-4.49 (m, 2H), 3.97-4.34 (m, 2H),
3.40-3.92 (m, 4H),
3.03-3.32(m, 6H), 2.52-2.85 (m, 4H), 2.19-2.45 (m, 3H), 2.03-2.17 (m, 3H),
1.83-1.95 (m, 1H),
1.70-1.79 (m, 111), 1.48-1.58 (m, 1H), 1.00-1.33 (m, 7H), 0.68-0.96 (m, 811).
Compound 49
111 NMR (400 MHz, CDCb) 6 ppm 8.48 (s, 1H), 7.52 - 7.40 (m, 1H), 7.39 - 7.31
(m, 211), 4.30
- 3.86 (m, 2H), 3.67 - 3.59 (m, 2H), 3.27 -2.97 (m, 9H), 2.94 -2.75 (m, 2H),
2.24 - 1.82 (m,
5H), 1.77 - 1.62 (m, 2H), 1.11 - 0.57 (m, 16H)
Compound 50
N1VIR (400 MHz, DMSO-d6) 6 ppm 8.48 (s, 1H), 7.40-7.48 (m, 1H), 7.29-7.39 (m,
2H),
3.47-4.31 (m, 9H), 3.24-3.45 (m, 3H), 2.92-3.12 (m, 3H), 1.93-2.22 (m, 4H),
1.50-1.92 (m, 3H),
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0.91-1.14 (m, 7H), 0.88 (d, J= 6.80 Hz, 3H), 0.83 (d, J= 6.40 Hz, 3H), 0.61-
0.79 (m, 2H).
Compound 51
1-1-1NMR_ (400 MHz, CDC13) 6 ppm 8.50 (s, 1H), 7.26 - 7.23 (m, 114), 7.17 -
7.10 (m, 1H), 7.07
- 6.99 (m, 1H), 4.84 - 4.75 (m, 2H), 4.46 -4.38 (m, 2H), 4.34 - 4.26 (m,
0.2H), 4.22 - 3.99 (m,
2H), 3.92 -3.82 (m, 0.8H), 3.80 -3.71 (m, 1H), 3.68 - 3.60 (m, 1H), 3.56 -
3.45 (m, 1H), 3.30
- 3.14 (m, 5H), 3.13 - 3.04 (m, 114), 2.82 -2.63 (m, 4H), 2.34 -2.23 (m, 214),
2.16 - 2.10 (m,
2H), 2.06 -2.02 (m, 1H), 1.97 - 1.85 (m, 1H), 1.80 - 1.67 (m, 3H), 1.12 - 1.02
(m, 614), 0.91 -
0.72 (m, 9H).
Compound 52
11-1 NMR (400 MHz, CDC13) 6 ppm 8.50 (s, 1H), 7.19-7.25 (m, 1H), 7.08-7.18 (m,
1H), 6.96-
7.08 (m, 1H), 4.71-4.89 (m, 2H), 4.38-4.48 (m, 2H), 3.44-4.34 (m, 6H), 3.01-
3.32 (m, 6H),
2.50-2.89 (m, 3H), 2.19-2.47 (m, 314), 2.04-2.18 (m, 3H), 1.83-1.95 (m, 1H),
1.70-1.77 (m, 1H),
1.62-1.70 (m, 1H), 1.50-1.60 (m, 1H), 1.01-1.27 (m, 7H), 0.72-0.92 (m, 8H).
Compound 78
IHNMIR CD3OD (Varian-400 MHz): 9.00 - 8.78 (m, 1H), 7.69 - 7.44 (m, 1H), 7.41 -
7.19 (m,
2H), 4.56 -4.13 (m, 6H), 4.05 - 3.78 (m, 2H), 3.58 (s, 1H), 3.49 - 3.32 (m,
5H), 3.13 - 2.97 (m,
2H), 2.72 -2.51 (m, 1H), 2.49 -2.31 (m, 1H), 2.23 - 1.91 (m, 4H), 1.89 - 1.65
(m, 214), 1.29 -
0.93 (m, 15H).
Compound 79
1H NMR CD3OD (Varian-400 MHz): 8.97 - 8.81 (m, 114), 7.72 -7.43 (m, 1H), 7.40 -
7.15 (m,
2H),4.61 - 4.15 (m, 6H), 4.08 - 3.70 (m, 2H), 3.58 (s, 1H),3.50 - 3.34 (m,
5H), 3.14 - 2.94 (m,
2H), 2.73 - 2.52 (m, 1H), 2.50 - 2.30 (m, 1H), 2.22- 1.89(m, 4H), 1.89-
1.64(m, 2H), 1.29 -
1.02 (m, 15H)
Compound 82:
1H -NIMR (400 MHz, CDC13): 8.55 - 8.41 (m, 114), 7.25 -7.18 (m, 11-1), 7.17 -
7.07 (m, 1H),
7.06 -6.95 (in, 1H), 4.78 ( t, J= 6.8 Hz, 2H), 4.39 ( t, J = 6.0 Hz, 2H), 4.31
- 3.95 (m, 2H), 3.93
- 3.81 (m, 1H), 3.78 - 3.42 (m, 3H), 3.39 - 2.99 (m, 514), 2.85 - 2.57 (m,
4H), 2.49 - 2.34 (m,
1H), 2.21 -2.00 (m, 3H), 1.91 - 1.70 (m, 1314), 1.49- 1.38 (m, 1H), 1.35- 1.22
(m, 1H), 1.18 -
0.69 (m, 8H).
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Compound 83:
1H NAIR (400 MHz, CDC13): 8.54- 8.39(m, 1H), 7.25 - 7.19(m, 1H), 7.13 (s, 1H),
7.06 - 6.97
(m, 1H), 4.78 ( t, J= 6.8 Hz, 2H), 4.39 ( t, 1= 6.2 Hz, 2H), 4.32 - 3.95 (m,
2H), 3.93 - 3.00 (m,
8H), 2.87 - 2.57 (m, 4H), 2.40 (s, 1H), 2.24 - 1.98 (m, 3H), 1.91 - 1.68 (m,
14H), 1.50 - 1.38
(m, 1H), 1.36- 1.22 (m, 111), 1.18 -0.67 (m, 8H).
Pi IARMACOLOGICAL PART
1) Menin/MLL homogenous time-resolved fluorescence (HTRF) assay
To an untreated, white 384-well microtiter plate was added 40 nL 200X test
compound in
DMSO and 4 [11_, 2X terbium chelate-labeled menin (vide infra for preparation)
in assay buffer
(40 mM Tris=HC1, pH 7.5, 50 mM NaCl, 1 mM DTT (dithiothreitol) and 0.05%
Pluronic F-
127). After incubation of test compound and terbium chelate-labeled menin for
30 min at
ambient temperature, 4 uL 2X FITC-MBM1 peptide (FTTC-13-alanine-SARWRFPARPGT-
NH2) ("FITC" means fluorescein isothiocyanate) in assay buffer was added, the
microtiter plate
centrifuged at 1000 rpm for 1 min and the assay mixtures incubated for 15 min
at ambient
temperature. The relative amount of menin=FITC-MBM1 complex present in an
assay mixture
is determined by measuring the homogenous time-resolved fluorescence (HTRF) of
the
terbium/FITC donor /acceptor fluorphore pair using an EnVision microplate
reader (ex. 337
nm/terbium em. 490 nm/FITC em. 520 nm) at ambient temperature. The degree of
fluorescence
resonance energy transfer (the HTRF value) is expressed as the ratio of the
fluorescence
emission intensities of the FITC and terbium fluorophores (Fern 520 nm/Fm 490
nm). The final
concentrations of reagents in the binding assay are 200 pM terbium chelate-
labeled menin, 75
nM FITC-MBM1 peptide and 0.5% DMSO in assay buffer. Dose-response titrations
of test
compounds are conducted using an 11 point, four-fold serial dilution scheme,
starting typically
at 10 M.
Compound potencies were determined by first calculating % inhibition at each
compound
concentration according to equation 1:
% inhibition = ((HC - LC) - (HTRFcompound LC)) / (HC - LC)) *100 (Eqn 1)
Where LC and HC are the HTRF values of the assay in the presence or absence of
a saturating
concentration of a compound that competes with FITC-MBM1 for binding to menin,
and
HTRF compound is the measured HTRF value in the presence of the test compound.
HC and LC
HTRF values represent an average of at least 10 replicates per plate. For each
test compound, %
inhibition values were plotted vs. the logarithm of the test compound
concentration, and the
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/C50 value derived from fitting these data to equation 2:
% inhibition = Bottom + (Top -Bottom)/(1+10^((log/C50-log[cmpd])*h)) (Eqn 2)
Where Bottom and Top are the lower and upper asymptotes of the dose-response
curve,
respectively, /C50 is the concentration of compound that yields 50% inhibition
of signal and h
is the Hill coefficient.
Preparation of Terbium cryptate labeling of Menin: Menin (a.a 1-610-6xhis tag,
2.3 mg/mL in
20mM Hepes (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethane sulfonic acid), 80 mM
NaCl, 5mM
DTT (Dithiothreitol), pH 7.5) was labeled with terbium cryptate as follows.
200 jig of Menin
was buffer exchanged into lx Hepes buffer. 6.67 tiM Menin was incubated with 8-
fold molar
excess NHS (N-hydroxysuccinimide)-terbium cryptate for 40 minutes at room
temperature.
Half of the labeled protein was purified away from free label by running the
reaction over a
NAPS column with elution buffer (0.1M Hepes, pH 7 + 0.1% BSA (bovine serum
albumin)).
The other half was eluted with 0.1M phosphate buffered saline (PBS), pH7. 400
j.tl of eluent
was collected for each, aliquoted and frozen at -80 C. The final concentration
of terbium-
labeled Menin protein was 115 ittg/mL in Hepes buffer and 85 ittg/mL in PBS
buffer,
respectively.
MENIN Protein Sequence (SEQ ID NO: 1):
MGLKAAQKTLFPLRSIDDVVRLFAAELGREEPDLVLLSLVLGFVEHFLAVNRVIPTNV
PELTF QP SPAPDPPGGLTYFPVADLSIIAALYARFTAQIRGAVDLSLYPREGGVS SREL
VKKVSDVIWNSLSRSYFKDRAHIQ SLF SF ITGTKLD SSGVAFAVVGACQALGLRDVH
LALSEDHAWVVFGPNGEQTAEVTWHGKGNEDRRGQTVNAGVAERSWLYLKGSYM
RCDRKIVIEVAFMVCAINP SIDLHTD SLELLQLQQKLLWLLYDLGHLERYPMALGNLA
DLEELEPTPGRPDPLTLYHKGIASAKTYYRDEHIYPYMYLAGYHCRNRNVREALQA
WADT A TVIQDYNYCREDEEIYKEF FEV ANDVIPNLLKE A A SLLEAGEERPGEQ S Q GT
Q S Q GS ALQDPECF AHLLRF YD GICKWEEGSP TPVLHVGWATFLVQ SLGRFEGQVRQK
VRIVSREAEAAEAEEPWGEEAREGRRRGPRRESKPEEPPPPKKPALDKGLGTGQGAV
SGPPRKPPGTVAGTARGPEGGSTAQVPAPAASPPPEGPVLTFQ SEKMKGMKELLVAT
KINSS A IKLQLTAQ S QVQMKK QK VS TP SDYTLSFLKRQRK GLEFITITHIH
2a) Proliferation assay
The anti-proliferative effect of menin/MLL protein/protein interaction
inhibitor test compounds
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was assessed in human leukemia cell lines. The cell line MOLM14 harbors a MLL
translocation and expresses the MILL fusion protein MILL-AF9, respectively, as
well as the
wildtype protein from the second allele. OCI-AMIL3 cells that carry the NPM1c
gene mutation
were also tested. MILL rearranged cell lines (e.g. M0LM14) and NPM1c mutated
cell lines
exhibit stem cell-like HOXA/MEIS1 gene expression signatures. KO-52 was used
as a control
cell line containing two MLL (KIVIT2A) wildtype alleles in order to exclude
compounds that
display general cytotoxic effects.
MOLM14 cells were cultured in RPMI-1640 (Sigma Aldrich) supplemented with 10%
heat-
inactivated fetal bovine serum (HyClone), 2 mM L-glutamine (Sigma Aldrich) and
50 g/m1
gentamycin (Gibco). KO-52 and OCI-AML3 cell lines were propagated in alpha-MEM
(Sigma
Aldrich) supplemented with 20% heat-inactivated fetal bovine serum (HyClone),
2 mM L-
glutamine (Sigma Aldrich) and 50pg/m1 gentamycin (Gibco). Cells were kept at
0.3 ¨ 2.5
million cells per ml during culturing and passage numbers did not exceed 20.
In order to assess the anti-proliferative effects, 200 MOLM14 cells, 200 OCI-
A1V1L3 cells or
300 KO-52 cells were seeded in 200u] media per well in 96-well round bottom,
ultra-low
attachment plates (Costar, catalogue number 7007). Cell seeding numbers were
chosen based
on growth curves to ensure linear growth throughout the experiment. Test
compounds were
added at different concentrations and the DMSO content was normalized to 0.3%.
Cells were
incubated for 8 days at 37 C and 5% CO2. Spheroid like growth was measured in
real-time by
live-cell imaging (IncuCyteZOOM, Essenbio, 4x objective) acquiring images at
day 8.
Confluence (%) as a measure of spheroid size was determined using an
integrated analysis tool.
In order to determine the effect of the test compounds over time, the
confluence in each well as
a measure of spheroid size, was calculated. Confluence of the highest dose of
a reference
compound was used as baseline for the LC (Low control) and the confluence of
DMSO treated
cells was used as 0% cytotoxicity (High Control, HC).
Absolute IC50 values were calculated as percent change in confluence as
follows:
LC = Low Control: cells treated with e.g. I ?AM of the
cytotoxic agent staurosporin, or
e.g. cells treated with a high concentration of an alternative reference
compound
HC = High Control: Mean confluence (%) (DMSO treated cells)
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% Effect = 100 - (100*(Sample-LC)/(HC-LC))
GraphPad Prism (version 7.00) was used to calculate the IC5o. Dose-response
equation was used
for the plot of % Effect vs Log10 compound concentration with a variable slope
and fixing the
maximum to 100% and the minimum to 0%.
2b) MEIS1 mRNA expression assay
1V1EIS1 mRNA expression upon treatment of compound was examined by Quantigene
Singleplex assay (Thermo Fisher Scientific). This technology allows for direct
quantification
of mRNA targets using probes hybridizing to defined target sequences of
interest and the signal
is detected using a Multimode plate reader Envision (PerkinElmer). The MOLM14
cell line
was used for this experiment. Cells were plated in 96-well plates at 3,750
cells/well in the
presence of increasing concentrations of compounds. After incubation of 48
hours with
compounds, cells were lysed in lysis buffer and incubated for 45 minutes at 55
C Cell lysates
were mixed with human MEIS1 specific capture probe or human RPL28 (Ribosomal
Protein
L28) specific probe as a normalization control, as well as blocking probes.
Cell lysates were
then transferred to the custom assay hybridization plate (Theimo Fisher
Scientific) and
incubated for 18 to 22 hours at 55 C. Subsequently, plates were washed to
remove unbound
materials followed by sequential addition of preamplifiers, amplifiers, and
label probe. Signals
(= gene counts) were measured with a Multimode plate reader Envision. IC5os
were calculated
by dose-response modelling using appropriate software. For all non-housekeeper
genes
response equal counts corrected for background and relative expression. For
each sample, each
test gene signal (background subtracted) was divided by the normalization gene
signal (RPL28:
background subtracted). Fold changes were calculated by dividing the
normalized values for
the treated samples by the normalized values for the DMSO treated sample. Fold
changes of
each target gene were used for the calculation of IC5os.
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Table 3. Biological data - HTRF assay, proliferation assay, and MEIS1 mRNA
expression
assay
HTRF- MEIS1 spheroid OCI- spheroid
Compound 30min ICso assay_OneTime AML3 assay_OneTime
Number incubation ( M) M0LM14 IC50 ICso KO-52
IC50
1050 (nM) (11,M) (111,M)
(IM)
1 1.03 >2.5
2 0.3 0.084 0.047 0.23 >15
3 8.3 1.89 1.81 >15
4 0.25 0.042 0.044 0.31
5.89
0.81 1.23 0.81 12.18
6 0.4 0.26 0.23 >15
7 8.11 >2.5 >3.75 >15
8 0.24 0.13 0.11 6.4
9 0.28 0.29 0.18
9.58
5.27 2.15 2.09 >15
11 0.28 0.19 0.26
11.67
12 3.61 >2.5 2.41 >15
13 2.1 1.64
14 0.16 0.18 0.14
13.57
2.44 1.51 0.8 >15
16 0.06 0.15 0.065 >15
17 0.25 0.14 0.11
4.02
18 0.65 0.66 0.6
11.75
19 0.39 0.14 0.071
11.89
0.14 >2.5
21 0.077 0.09 0.068
10.67
22 0.18 0.049 0.043 0.57
14.69
24 0.095 0.01 0.009
12.18
2.28 0.69 0.63 >15
27 0.074 0.025 0.009
7.93
29 1.94 0.65 0.21 >15
2.6 0.33 0.22 13.33
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HTRF- MEIS1 spheroid OCI- spheroid
Compound 30min IC50 assay_OneTime AML3 assay_OneTime
Number incubation ( 1V1) MOLM14 IC5o ICso KO-52
IC50
IC50 (nM) (111M) (PM)
(1[11V1)
31 0.18 0.032 0.02 13
34 0.056 0.48
35 1.24 >2.5
38 0.62 0.7 1.05 >15
39 0.61 0.12 0.11
6.09
40 6.23 >2.5 1.85 >15
41 0.097 0.052 0.04
9.06
42 0.2 0.15 0.1
9.76
43 0.049 0.13 0.089 0.48
8.32
44 2.72 1.88
45 0.64 >2.5
46 0.29 1.31
47 2.26 1.25
48 0.16 0.19
49 0.58 1.48
50 0.39 >2.5
51 0.2 0.19 0.23
11.04
52 0.73 1.21 0.96 >15
53 0.13 0.38 0.43 >15
55 0.17 0.0014 0.0018 0.012
13.59
56 0.056
57 0.064 0.002 0.0018 0.0085
3.82
58 0.21 0.051 0.038 0.16
7.17
59 0.29 0.014 0.012 0.081
4.88
60 36.38
61 0.15 <0.0034 0.006 0.022
7.87
62 1.21 0.038
63 3.03 0.38
64 1.97 >1
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HTRF- MEIS1 spheroid OCI- spheroid
Compound 30min IC50 assay_OneTime AML3 assay_OneTime
Number incubation ( 1V1) MOLM14 IC5o ICso KO-52
IC50
IC50 (nM) (111M) (PM)
(1[11V1)
65 0.074 0.0066 0.012 0.057
1.7
66 0.21 0.027 0.16
67 1.65
68 3.69
69 0.073 0.0052 0.012 0.067
1.6
70 0.23 0.064 0.043 0.73 >15
71 0.13 0.13 0.08 2
72 0.22 0.18 0.08
2.2
73 0.21 0.11 0.08
6.34
74 0.4 0.093 0.048 0.26
9.06
75 1.12 0.71 1.13
10.83
78 2.73 >2.5 >3.75 >15
79 0.067 0.64 0.73 >15
82 0.3 0.11 0.068 0.34
8.53
83 0.35 0.29 0.21
9.24
84 0.16 0.12 0.056 >15
85 2.84 2.47 0.89 >15
86 0.19 0.28 0.11 >15
87 2.12 >2.5 1.59 >15
88 7.51 >2.5 1.78 >15
89 0.42 0.25 0.1 >15
90 0.38 0.2 0.17 >15
91 0.24 0.15
7.61
92 101.23 >2.5 >3.75 >15
93 1.09 0.51 1.1 >15
94 0.44 0.32 0.49 >15
95 0.92 0.41 0.5
6.12
96 2.35 0.51 0.55
10.58
97 2.45 0.73 1.41 >15
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HTRF- MEIS1 spheroid OCI- spheroid
Compound 30min IC50 assay_OneTime AML3 assay_OneTime
Number incubation ( 1V1) MOLM14 IC5o ICso KO-
52 IC50
IC50 (nM) (111M) (PM)
(1[11V1)
98 0.71 0.26 0.19
8.46
99 1.46 0.11
6.08
100 1.49 1.33 >15
101 0.68 0.33 0.31 >15
102 0.49 0.47 >15
103 0.25 0.25 0.17 >15
104 8.91 2.08 3.28 >15
105 0.49 0.096 0.087
12.52
106 0.57 1.45 1.1 >15
107 0.38 0.25 0.14
12.56
108 0.55 0.38 0.18
9.32
109 1.65 0.63 0.27
5.91
110 1.5 0.68 0.22
14.56
111 0.25 0.2 0.03 >15
112 0.56 >1 >0.94 9.64 >15
113 0.25 0.48 0.34 1.41 >15
114 0.12 0.048 0.029 0.14
12.61
115 0.97 0.58 0.77 2.48
15.67
116 0.34 >0.94 1.32
13.29
117 0.89 0.4 0.57 1.89
6.67
118 0.048 0.13 0.32
9.55
119 0.9 0.41 0.47
13.1
120 0.093 0.0046 0.0053 0.035
121 0.44 0.17 0.091 1.16
122 0.36 0.13 0.13 6.4
123 0.35 0.17 0.22
5.89
124 0.38 0.14 0.12
14.6
125 0.22 0.16 0.13
11.35
126 0.4 0.19 0.095
11.54
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HTRF- MEIS1 spheroid OCI- spheroid
Compound 30min IC5o assay_OneTime AML3 assay_OneTime
Number incubation ( 1V1) MOLM14 IC5o IC5o KO-52
IC5o
IC50 (nM) (111M) (PM)
(11M)
127 0.26 0.032 0.0058 0.17
128 0.1 0.027 0.029 0.075
129 0.066 0.0044 0.006 0.049
131 0.22 0.0052 0.007 0.026
132 0.68
134 0.18 0.037
135 0.15 0.026
136 2.2 0.27
137 0.45 0.39 0.37 >15
139 3.07 0.91
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