NZ719122B2 - Processes for making compounds useful as inhibitors of ATR kinase - Google Patents
Processes for making compounds useful as inhibitors of ATR kinase Download PDFInfo
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- NZ719122B2 NZ719122B2 NZ719122A NZ71912212A NZ719122B2 NZ 719122 B2 NZ719122 B2 NZ 719122B2 NZ 719122 A NZ719122 A NZ 719122A NZ 71912212 A NZ71912212 A NZ 71912212A NZ 719122 B2 NZ719122 B2 NZ 719122B2
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- New Zealand
- Prior art keywords
- compound
- mmol
- hcl
- tert
- deuterium
- Prior art date
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- 230000001131 transforming Effects 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/4965—Non-condensed pyrazines
- A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C251/32—Oximes
- C07C251/34—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
- C07C251/48—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with the carbon atom of at least one of the oxyimino groups bound to a carbon atom of a six-membered aromatic ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C269/06—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups by reactions not involving the formation of carbamate groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
- C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
- C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D241/20—Nitrogen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
- C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
- C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D309/14—Nitrogen atoms not forming part of a nitro radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
Abstract
The present disclosure relates to processes and intermediates for preparing compounds useful as inhibitors of ATR kinase, such as aminopyrazine-isoxazole derivatives and related molecules. The present disclosure also relates to compounds useful as inhibitors of ATR protein kinase. The disclosure relates to pharmaceutically acceptable compositions comprising the compounds; methods of treating of various diseases, disorders, and conditions using the compounds; processes for preparing the compounds; intermediates for the preparation of the compounds; and solid forms of the compounds. The compounds have formula (I) or (II) wherein the variables are as defined herein. ates to pharmaceutically acceptable compositions comprising the compounds; methods of treating of various diseases, disorders, and conditions using the compounds; processes for preparing the compounds; intermediates for the preparation of the compounds; and solid forms of the compounds. The compounds have formula (I) or (II) wherein the variables are as defined herein.
Description
PATENTS FORM NO. 5 Our ref: JX 237282NZPR
DIVISIONAL APPLICATION FILED OUT OF NZ 623089
NEW ZEALAND
PATENTS ACT 1953
COMPLETE ICATION
Processes for making compounds useful as inhibitors of ATR kinase
We, Vertex Pharmaceuticals Incorporated of 50 Northern Avenue, Boston, 02210,
Massachusetts, United States of America hereby declare the invention, for which we pray that a
patent may be d to us and the method by which it is to be performed, to be particularly
described in and by the following statement:
(Followed by page 1a)
103831200_1.docx:JX:ewa
VPI/11-127 WO
PROCESSES FOR MAKING COMPOUNDS USEFUL AS TORS OF ATR KINASE
BACKGROUND OF THE INVENTION
ATR (“ATM and Rad3 related”) kinase is a protein kinase involved in cellular
responses to DNA damage. ATR kinase acts with ATM (“ataxia telangiectasia mutated”) kinase
and many other proteins to regulate a cell’s response to DNA damage, commonly referred to as
the DNA Damage se (“DDR”). The DDR stimulates DNA repair, promotes survival and
stalls cell cycle progression by activating cell cycle checkpoints, which e time for repair.
Without the DDR, cells are much more sensitive to DNA damage and readily die from DNA
lesions d by endogenous ar processes such as DNA replication or exogenous DNA
damaging agents commonly used in cancer therapy.
Healthy cells can rely on a host of different proteins for DNA repair including the
DDR kinase ATR. In some cases these ns can compensate for one another by activating
functionally redundant DNA repair processes. On the contrary, many cancer cells r
defects in some of their DNA repair ses, such as ATM signaling, and therefore display a
greater reliance on their remaining intact DNA repair proteins which include ATR.
In addition, many cancer cells express activated oncogenes or lack key tumour
suppressors, and this can make these cancer cells prone to dysregulated phases of DNA
replication which in turn cause DNA damage. ATR has been implicated as a critical ent
of the DDR in response to disrupted DNA replication. As a result, these cancer cells are more
dependent on ATR activity for al than healthy cells. Accordingly, ATR inhibitors may be
useful for cancer treatment, either used alone or in combination with DNA damaging agents,
because they shut down a DNA repair mechanism that is more important for cellular survival in
many cancer cells than in healthy normal cells.
In fact, tion of ATR function (e.g. by gene deletion) has been shown to
promote cancer cell death both in the absence and presence of DNA damaging agents. This
suggests that ATR inhibitors may be effective both as single agents and as potent sensitizers to
radiotherapy or genotoxic chemotherapy.
(Followed by page 2)
103831200_1.docx:JX:ewa
PCT/U82012/058127
For all of these reasons, there is a need for the development of potent and selective
ATR inhibitors for the treatment of cancer, either as single agents or as combination therapies
with radiotherapy or genotoxic chemotherapy. Furthermore, it would be desirable to have a
tic route to ATR inhibitors that is amenable to large—scale synthesis and improves upon
currently known methods.
ATR peptide can be expressed and isolated using a y of methods known in
the literature (sgg e. g., Unsal-Kacmaz et al, PNAS 99: 10, pp6673-6678, May 14, 2002; see
al_so Kumagai et a1. gel; 124, pp943-955, March 10, 2006; Unsal—Kacmaz et al. Molecular
and ar Biology, Feb 2004, p1292-1300; and Hall—Jackson et a1. Oncogene 1999, 18,
67076713).
DESCRIPTION OF THE FIGURES
FIGURE 1a: XRPD Compound I—2 free base
FIGURE 2a: TGA Compound I-2 free base
FIGURE 3a: DSC Compound 1-2 free base
FIGURE 43: ORTEP plot ofthe asymmetric unit of the Compound 1-2 free form single
crystal structure
FIGURE 1b:XRPD nd 1-2 0 HCl
FIGURE 2b: TGA Compound 1-2 ° HCl
FIGURE 3b: DSC Compound I—2 ' HCl
FIGURE 4b: ORTEP plot of the asymmetric unit of the nd 1—2 ° HCl anhydrous
structure.
FIGURE 10: XRPD Compound 1—2 - 2HC1
FIGURE 2c: TGA Compound 1—2 ° 2HC1
FIGURE 30: DSC Compound I—2 ° 2HC1
FIGURE 1d: XRPD Compound 1—2 - HCl monohydrate
FIGURE 2d: TGA nd 1—2 ° HCl monohydrate
FIGURE 3d: DSC Compound I-2 ° HCl monohydrate
FIGURE 1e: XRPD Compound I—2 - HCl * 21-120
FIGURE 2e: TGA Compound 1—2 ° HCl - 2H20
FIGURE 3e: DSC Compound I-2 - HCI ° 2H20
FIGURE 4a: Solid State Compound I—l free base
FIGURE 4b: Solid State 13CNMR of Compound I-l - HCl
W0 2013f049726 PCT/U52012/058127
SUMMARY OF THE INVENTION
The present invention relates to processes and intermediates for preparing
compounds useful as inhibitors of ATR kinase, such as aminopyrazine-isoxazole tives
and related molecules. Aminopyrazine—isoxazole tives are useful as ATR inhibitors and
are also useful for preparing ATR inhibitors. The present invention also s to solid forms
of ATR inhibitors as well as deutcrated ATR inhibitors.
One aspect of the invention provides a process for preparing a compound of
formula 1:
NH o’N 3
\\ / WN’R
NO +3 H
YN J1
sing preparing a compound of formula 4:
HO-N\ / ”NR3
—l=/ FI’G
from a compound of formula 3:
Rl—O
R2_O Lit] '56
under suitable oxime formation conditions.
Another aspect comprises preparing a compound of formula 4:
HO"N\ / WN’R3
’J FI’G
from a compound of formula 3:
/ WNIRB
R2_O -|:/ [56
under suitable oxime formation conditions.
PCT/U82012/058127
Another aspect of the present invention comprises a compound of formula II:
R4 R33
0\ R3b
0// R30
R1 2 R1 0
R1 0
or a pharmaceutically acceptable salt thereof,wherein each R1“, Rlb, R”, R2, R33, R3b,
R3“, R4, R5, R6, R7, R8, R93, R91), R10, R11, R12, and R13 is independently hydrogen or
deuterium, andat least one of R”, R‘b, R”, R2, R3“, R3b, R32 R4, R5, R6, R7, R8, R93, R911 R10,
R“, R”, and R13 is deuterium.
Yet another aspect of the invention provides solid forms of a compound of
formula 1-2:
NH2 o—“l HN‘
0:8CO
Other aspects of the invention are set forth .
The present invention has several advantages over previously known methods.
First, the present process has fewer number of total tic steps compared with previously
disclosed ses. Second, the present process has improved yields over previously
disclosed ses. Third, the present process is effective for compounds wherein R3 is a
wide range of groups, such as alkyl groups or a large, hindered , such as a ring. Fourth,
W0 2013IO49726 PCT/U52012/058127
the present process ses intermediates which are more stable and have a longer shelf
life. In certain embodiments, the non-acidic formation of the oxime group in the present
process allows the preservation of acid-sensitive protecting groups such as Boc or CB2
during the course of the synthesis. In other embodiments, the process is more easily scaled up
to larger quantities due to the elimination of chromatography as a purification step.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of the invention provides a process for making a compound of
preparing a compound of a 4:
HO-—N R3
\ / ”N,
-l=/ FI’G
from a compound of formula 3:
R1—o R3
/ ”N,
R2_O -|:/ I'l’G
under suitable oxime formation ions;
wherein
R1 is C1_6alkyl;
R2 is C1_6alkyl;
or R1 and R2, together With the oxygen atoms to which they are attached, form an
optionally substituted 5 or 6 membered saturated heterocyclic ring having two oxygen
atoms;
R3 is hydrogen, C\1_6alkyl, or a 3—6 membered saturated or partially unsaturated
cyclyl having 1-2 heteroatoms selected from the group consisting of oxygen,
nitrogen, and sulfur; wherein the heterocyclyl is optionally substituted with l
occurrence of halo or ky1;
J'1 is halo, C14alkyl, or CMalkoxy;
PG is a atc protecting group.
Another aspect provides a process for ing a compound of formula I:
WO 49726
comprising the steps of:
preparing a compound of formula 4:
HO—N ,R3
\ / ”N
from a compound of formula 3:
under suitable oximc formation ions;
R1 is Cmalkyl;
R2 is C1.6alky1;
or R1 and R2, together with the oxygen atoms to which they are attached, form an
Optionally tuted 5 or 6 membered saturated heterocyclic ring having two oxygen
atoms;
R3 is hydrogen, C~1_6alkyl, or a 3—6 membered saturated or partially unsaturated
heterocyclyl having 1—2 heteroatoms selected from the group consisting of oxygen,
nitrogen, and sulfur; wherein the heterocyclyl is optionally substituted with l
occurrence of halo or C1_3alkyl;
(J2)q
R4 is ;
Q is phenyl, pyridyl, or an N—alkylated pyridine;
J‘ is 1-1, halo, CMalkyl, or C1.4a1koxy;
PCT/U52012/058127
12 is halo; CN; ; oxazolyl; or a C1.5aliphatic group wherein up to 2 methylene units
are optionally replaced with 0, NR”, C(O), S, 8(0), or 8(0):; said C1.5aliphatic group
is optionally substituted with 1~3 fluoro or CN;
q is 0, 1, or 2;
PG is a carbamate protecting group.
Another embodiment further comprises the step of protecting a compound of
formula 2:
R1-O ,R3
RZ-O ‘i—
under suitable protection ions to form the compound of formula 3.
Another embodiment further comprises the step of reacting a compound of
formula 1:
R1-O
/ W0
with a suitable amine under suitable reductive amination conditions to form a compound of
formula 2.
In some embodiments, the suitableamine is NHCH3. In other embodiments, the
suitable amine IS .
Another embodiment further comprises the step of reacting a nd of
formula 4:
under suitable isoxazole formation conditions to form a nd of formula 5:
Another ment further comprises the step of reacting a nd of
formula 5 under suitable coupling conditions followed by suitable deprotection conditions to
form a compound of formula I.
In some embodiments, PG is Boc or Cbz. In some embodiments, PG is Boc.
In other embodiments, R1 is ethyl and R2 ethyl.
In yet other embodiments, R3 is CH3 or é—C .
02):;
In some embodiments, R4 is ; wherein Q is phenyl. In some
embodiments, Q is substituted in the para position with J2, wherein q is 1.
In some embodiments, J1 is H or halo. In some embodiments, J1 is H. In other
embodiments, J is halo.
In other ments, J2 is a C1_6aliphatic group wherein up to l methylene unit
is ally replaced with S(O)2. In some embodiments, J2 is ~S(O)2—(C1_5alkyl). In some
embodiments, q is 1.
According to another embodiment,
R1 is ethyl;
R2 is ethyl;
R3 is CH3 or $.00;
PG is Boc or Cbz;
J] is H;
02%.
R4 is wherein Q is phenyl; J2 is —S(O)2-CH(CH3)2;
q is 1;
2012/058127
In some embodiments, R3 is CH3. In some ments, R3 is CH3. In yet
E 0
another embodiments, R3 is CH3 or ‘C .
According to another embodiment,
R1 is ethyl;
R2 is ethyl;
a s0
R” is '
PG is B00;
1‘ is H;
(an 3X
R4 is wherein Q is pyridyl; J2 is N
q is 1;
\ CH3
N CH3
In some embodiments, R_ 4 _
IS N
Reactions Conditions
In some embodiments, the suitable oxime ion conditions consist of either
a single step sequence or a two step ce.
In some embodiments, the two step sequence consists of first deprotecting the
ketal group in the nd of a 3 into an aldehyde under suitable deprotection
conditions, and then forming the oxime of formula 4 under suitable oxime formation
conditions. In some embodiments, suitable deprotcction conditions comprise adding catalytic
amounts of para—toluenesulfonic acid (pTSA), acetone, and water; and suitable oxime
formation conditions comprise mixing together hydroxylamine, a catalytic amount of acid, a
dehydrating agent, and an alcoholic solvent. In other embodiments, the acid is pTSA or HCl,
the dehydrating agent is molecular sieves or dimethoxyacetone, and the alcoholic solvent is
ol or ethanol.
In other embodiments, the single step sequence comprises adding NHzOH.HCl
and a mixture of THF and water. In other embodiments, the sequence comprises adding
PCT/USZ012/058127
NHzOHHCl with a mixture of 2-methyl tetrahydrofuran and water optionally buffered with
Na2804. In some embodiments, 1 equivalent of the compound of formula 3 is ed with
a 1.1 equivalents ofNHZOHHCl in a 10:1 v/v mixture ofTHF and water. In some
embodiments, 1 equivalent of the nd of formula 3 is combined with a 1.1 equivalents
ofNHZOHHCI in a 10:1 v/v mixture of 2—methyl tetrahydrofuran and water optionally
buffered with NaZSO4.
In other embodiments, the protection ions are selected from the group
consisting of
o R—OCOCl, a suitable ry amine base, and a suitable solvent; wherein R is
C1_5alkyl optionally substituted with phenyl;
o R(C02)OR’, a le solvent, and optionally a catalytic amount of base, wherein
R is and R’ are each independently C1.6alkyl optionally substituted with phenyl;
0 [RO(C=O)]20, a suitable base, and a suitable t.
In some embodiments, the suitable base is Et3N, diisopropylamine, and pyridine;
and the suitable solvent is selected from a chlorinated solvent, an ether, or an aromatic
arbon. In other embodiments, the le base is Eth, the suitable solvent is a
chlorinated solvent selected from DCM. In yet other embodiments, the tion conditions
se adding 1.20 lents of (Boe)20 and 1.02 equivalents of Eth in DCM.
According to r embodiment suitable coupling conditions comprise adding
a suitable metal and a suitable base in a suitable solvent. In other embodiments, the suitable
metal is Pd[P(tBu)3]2; the suitable solvent is a mixture of acetonitrile and water; and the
suitable base is sodium carbonate. In yet other embodiments, the suitable coupling conditions
comprise adding 0.1 equivalents of Pd[P(tBu)3]2; 1 equivalent of boronic acid or ester; and 2
equivalents of sodium carbonate in a 2:1 ratio v/v of acetonitrile/watcr at 60-70°C.
According to another embodiment, suitable deprotection conditions comprise
combining the compound of formula Q with a suitable acid in a suitable solvent. In some
embodiments, the suitable acid is selected from para-toluenesulfonic acid , HC],
TBAF, H3PO4, or TFA and the suitable solvent is ed from acetone, methanol, ethanol,
, EtOAc, THF, F, dioxane, toluene, or diethylether.
According to another embodiment, suitable isoxazole-formation conditions
consists of two steps, the first step comprising reacting the compound of formula 4 under
suitable chlorooxime formation conditions to form a chlorooxime intermediate; the second
step comprising reacting the chlorooxime intermediate with acetylene under suitable
cycloaddition conditions to form a compound of a 5.
According to another embodiment, suitable chlorooxime formation conditions are
selected from
. N—chlorosuccinimide and suitable solvent or
' potassium peroxymonosulfate, HCl, and dioxane.
In some embodiments, the suitable solvent is selected from a nonprotic solvent, an
aromatic hydrocarbon, or an alkyl acetate. According to another embodiment, the suitable
chlorooximc formation ions are 1.05 equivalents ofN—chlorosuccinimide in
isopropylacetate at 40-50°C.
According to another embodiment, suitable cycloaddition conditions consist of a
suitable base and a suitable solvent. In some embodiments, the suitable base is selected from
pyridine, DIEA, TEA, t-BuONa, and K2C03 and the suitable solvent is selected from
acetonitrile, tetrahydrofuran, 2—methyltetrahydrofuran, MTBE, EtOAc, i—PrOAc, DCM,
toluene, DMF, and ol. In other embodiments, the suitable base is selected-from Et3N
and the suitable solvent is selected from DCM.
According to another embodiment, the second step comprises reacting 1
equivalent of ene with 1.2 lents of the chlorooxime ediate and 1.3
equivalents ofRN in DCM at room temperature.
ing to another embodiment, suitable isoxazoIe-formation conditions
comprise ing the compound of formula 4 with an oxidant in a suitable solvent. In som
embodiments, said oxidant is [bis(trifluoroacetoxy)iodo] benzene and said solvent is a 1:121
mixture of ol, water, and dioxane.
Synthesis of Compounds I—2 and L3
One embodiment provides a process for preparing a compound of a 1-2:
NH2 0"‘{ HN\
SOziPr
sing one or more of the following steps:
PCT/USZ012/058127
a) Reacting a compound of formula 1b:
EC 3 0
EC H
with methylamine under suitable ive amination ions to form a compound of
formula 2b:
2b .
b) reacting a compound of formula 2b under suitable Boc protection conditions to
form the compound of formula 3b:
EtOJ\©\/{[300N\
3b .
c) reacting a compound of formula 3b under suitable oxime formation conditions to
form the nd of a 4-i:
HOc|N
[1300
d) reacting a compound of formula 4-i under suitable chlorooxime formation
conditions to form the compound of formula 4—ii:
4-ii
e) reacting the compound of formula 4—ii with a compound of formula 4—iii
W0 2013I049726 PCTIU52012/058127
f) .reacting a compound of formula 4-iV with a compound of formula A—S—i:
B(OH)2
i-PrOZS
A-S-i
under suitable coupling conditions to form the compound of formula 5-i:
Boos ’Boc 300‘
N 0 N
SOZiPr
g) deprotecting a compound of formula 5—i under le Boc deprotection
conditions optionally ed by treatment under basic aqueous conditions to
form a compound of formula 1-2.
Another embodiment provides a process for preparing a compound of formula
1-3:
2012/058127
SOgiPr
comprising one or more of the following steps:
a) Reacting a compound of formula A—l:
EC 8 O
HO H
with tetrahydro-2H-pyran-4—amine under suitable reductive amination conditions to form a
compound of formula A-2:
EC 3 HN‘CO
b) reacting a compound of formula A-2 under suitable Boc protection conditions to
form the compound of formula A—3:
‘ Boc
c) reacting a nd of formula A—3 under suitable oxime formation ions to
form the compound of formula A-4:
d) reacting a compound of formula A-4:
W0 2013f049726 PCT/U82012/058127
N“$69
under suitable oxime formation conditions to form the compound of formula
Ai:
HO\ O
“*CgN‘BocN
A—4-i
e) reacting the compound of a Ai with a compound of formula A—4—ii:
N(BOC)2
‘5»:
Aii
under suitable cycloaddition conditions to form the compound of formula A-S:
Boc\ /
N \ W”N l N\Boc
Br (j0
f) reacting a compound of formula A-S with a nd of formula A—5-i:
i-PrOZS
Ai
under suitable coupling conditions to form the compound of formula A-6:
PCT/U82012/058127
SOziPr
g) deprotecting a compound of formula A-6 under suitable Boc deprotection
conditions optionally followed by treatment under basic aqueous conditions to
form a compound of formula 1-3.
Suitable coupling conditions comprise combining a suitable palladium catalyst
with a le base in a suitable solvent. Suitable palladium catalyst include, but are not
d to, Pd[P(tBU)3]2, Pd(dtbpf)C12, Pd(PPh3)2Clz, Pd(PCy3)2Clg and
, Pd(dppf)C12,
Pd(dppe)C12. Suitable solvents include, but are not limited to. toluene, MeCN, water, EtOH,
IPA, 2—Me—THF, or IPAc. Suitable bases include, but are not limited to, K2CO3, Na2CO3, or
K3PO4.
Suitable oxime ion conditions consist of either a single step ce or a
two step ce. The two step sequence consists of first deprotecting the ketal group in the
compound of formula A—3 into an de under suitable deprotection conditions, and then
forming the oxime of formula A-4 under suitable oxime formation conditions.
The single step ce comprises, for example, se mixing together
hydroxylamine, an acid, an organic solvent, and water. In some embodiments, NH20H.HC1 is
added to a mixture of THF and water. In some embodiments, 1 lent of the compound
of formula 3-A is combined with a 1.1 equivalents ofNHgOHHCl in a 10:1 v/v mixture of
THF/water.
Suitable deprotection conditions comprise adding an acid, acetone, and water.
Suitable acids include pTSA or HCl, tsuitable organic solvents include chlorinated solvents
(e. g., dichloromethane (DCM), dichloroethane (DCE), CHZClz, and chloroform); an ether
(e.g., THF, 2—MeTHF and dioxane); an ic hydrocarbons (e. g., toluene and xylenes, or
other c solvents.
PCT/U52012/058127
Suitable cycloaddition ions comprise a suitable base (e.g., pyridine,
DIEA, TEA, a, or K2C03) and a suitable solvent (e.g., acetonitrile, tetrahydrofuran,
2-methyltetrahydrofuran, MTBE, EtOAc, i-PrOAc, DCM, toluene, DMF, and methanol_.
le chlorooxime formation conditions comprise adding HCl in dioxame to
a solution of the oxime in the presence ofNOS in a suitable solvent ed from a nonprotie
solvents (DCM, DCE, THF, and dioxane), aromatic hydrocarbons (e.g. toluene, xylenes), and
alkyl acetates (e.g., isopropyl acetate, ethyl acetate).
Suitable Boc deproteetion conditions comprises adding a suitable Boc
deproteeting agent (e. g, TMS—Cl, HCl, TBAF, H3PO4, or TFA) and a suitable solvent (e.g.,
acetone, toluene, methanol, ethanol, l-propanol, isopropanol, CHZCIZ, EtOAc, isopropyl
acetate, tetrahydrofuran, 2~methyltetraydrofuran, dioxane, and diethylether). In some
embodiments, the suitable Boc deprotection conditions comprises adding a suitable Boc
deprotecting agent selected from HCl, TFA and a suitable solvent selected from acetone,
toluene, isopropyl acetate, tetrahydrofuran, or 2—methyltetraydrofuran.
Suitable Boc protection conditions include 0, a suitable base, and a
suitable solvent. Suitable bases e, but are not limited to, Eth, diisopropylamine, and
pyridine. le solvents include, but are not limited to, chlorinated solvents (e. g.,
diehloromethane (DCM), dichloroethane (DCE), CHzClz, and chloroform); an ether (e. g.,
THF, 2-MeTHF and dioxane); an aromatic hydrocarbons (e.g., toluene and xylenes, or other
aprotic solvents. In some embodiments, the suitable base is Eth, the suitable solvent is
DCM, tetrahydrofuran or 2—methy1tetrahydrofuran. In certain embodiments, the protection
conditions comprise adding 105 equivalents of (Boc)20 in 2~methyltetrahydrofi1ran or DCM.
le reductive amination conditions comprise adding a ng agent
selected from NaBH4 NaBH4, NaBH3CN, or NaBI-I(OAC)3 in the presence of a solvent
selected from diehloromethane (DCM), diehloroethane (DCE), an alcoholic solvent selected
from methanol, ethanol, 1~propanol, isopropanol, or a nonprotie solvent selected from
dioxane, tetrahydrofuran, or yltetrahydrofuran and optionally a base ed from
Eth or diisopropylethylamine. In some embodiments, the suitable reductive amination
conditions se adding 1.2 lents 4 caplets in the presence Et3N in MeOH.
Another aspect of the present invention provides a compound of Formula II:
PCT/USZ012/058127
R4 R33
O§S R3b
0// R30
R13 R10
or a pharmaceutically acceptable salt thereof,
. 7
Whereln e3,011 R13, Rlb, Ric, R", R38, R31), R30, R4, R5, R6, R7, R8, R91, Rgb, R103, RIOb,(
and R100 is independently hydrogen or deuterium, and
at least one of Rla’ Rib, Rlc’ R2, R33, R31»’ R3c, R4, R5, R6, R7, R8, R9“, Rm), RIOa’ R1015,
and R10c is deuterium.
In some embodiments, R98 and R91) are the same. In other embodiments, R93 and
R9b are deuterium, and R”, R“: R‘“, R2, R33, R3“, R3“, R4, R5, R6, R7, R8, R10, R' "d, R‘ “1 Rn“,
Rm, R13“, Rm), R143, and R14b are deuterium or hydrogen. In yet another embodiment, Rga
and R9b are deuterium, and R”, R“: R1“, R2, R3”: R3b, R32 R4, R5, R6, R7, R8, R‘”, 12““, R1”,
R123, Rub, R133, Rm’, R14“, and R1419 are hydrogen.
In one embodiment, R9“, R91), R10“, Rum, and Rmc are the same. In another
embodiment, Rga, Rgb, R103, Rmb, and Rmc are deuterium, and R1“, Rib, R1“, R2, R3“, R3b, Rh,
R4, R5, R6, R7, and R8 are ium or hydrogen. In some embodiments, R9”, R9b, Rwa, Rmb,
and R10: are deuterium, and R”, R“), R“, R2, R3a, R3b, R3°, R4, R5, R6, R7, and R8 are
hydrogen.
In other embodiments, R10“, wa, and R10c are the same. In one embodiment,
R103, Rmb, and R1°° are deuterium, and R“, Rm, R”, R2, R3“, R”, R3“, R4, R5, R6, R7, R8, R93,
and R9b are ium or hydrogen. In yet another embodiment, R10“, Rmb, and Rloc are
ium, and R13, R“), R”, R2, R33, R”, R“, R4, R5, R6, R7, R8, R9“, and Rgb are hydrogen.
In some embodiments, R”, Ru’, R”, R2, R38, R3b, and R3° are the same. In
another embodiment R”, R”, R1“, R2, R3”, R3“, and R3c are deuterium, and R4, R5, R6, R7, R8,
Rga, R9b, R103, R101”, and R10C are deuterium or en. In yet another embodiment, R”, R”,
PCT/U52012/058127
R‘“, R2, R33, R3”, and R36 are deuterium, and R4, R5, R6, R7, R8, R9a, RQ", Rm”, Rm", and R10“
are deuterium.
In another embodiment, R6 is deuterium, and R13, Rlb, R“, R2, R3“, R33, R3°, R4,
R5, R7, R8, R93, R9b, R103, Rlob, and R10c are deuterium or hydrogen. In yet another
embodiment, R6 is ium, and R13, R”, R”, R2, R38, R“: R3“, R4, R5, R7, R8, R93, 119‘“,
Rloa, RIOb, and R10c are hydrogen.
In other embodiments, R2 is deuterium, and R”, Rlb, R1“, R38, R3b, and R3°, R4,
R5, R6, R7, R8, R9“, R913, R103, Km, and R10c are deuterium or hydrogen. In another
ment, R2 is deuterium, and R18, R”, R”, R3“, R31”, and R3“, R4, R5, R6, R7, R8, R93, R91”,
Rm", Rlob, and R10c are hydrogen.
In another embodiment, R7 is deuterium, and R18, R”, R”, R2, R33, RSb, R3°, R4,
R5, R6, R8, R93, R91”, Rica, Rum, and R100 are deuterium or hydrogen. In other ments,
R7 is deuterium, and R13, Rlb, R10, R2, R33, R”, R36, R4, 115,116, R8, R9“, Rgb, R103, R10”, R‘°°
are hydrogen.
In yet another embodiment, R8 is ium, and R”, R”, R”, R2, R3“, R312 R3”,
R4, R5, R6, R7, R93, Rgb, R108, Rmb, and R10c are deuterium or hydrogen. In another
embodiment, R8 is deuterium, and R13, R“: R“, R2, R3“, R3b, R32 R4, R5, R6, R7, R9“, R9b,
Rm“, R‘Ob, R10c are hydrogen.
In some embodiments, at least one of Rm“, Rmb, or R10C are the same. In
another embodiment, at least one of R103, Rmb, or ch are deuterium, and R13, R”, R”, R2,
R33, R“: R3”, R4, R5, R6, R7, R8, R93, and R9b are deuterium or en. In yet another
embodiment, at least one of Rm”, Rm", or R10C are deuterium, and R”, R”, R”, R2, R3”, R3”,
R3“, R4, R5, R6, R7, R8, R93, and R91) are hydrogen.
In some embodiments, at least two of R103, Rmb, or R10c are the same. In
another embodiment, at least two of Rloa, R1011 or R10c are deuterium, and R”, R”, R”, R2,
R“, R”, R3°, R4, R5, R6, R7, R8, R98, and R91) are deuterium or hydrogen. In yet another
embodiment, at least two of R103, R”, or R“)c are deuterium, and R13, R”, R”, R2, R33, R”,
RR, R4, R5, R6, R7, R8, R93, and R91) are hydrogen.
In another embodiment, R13, R”, RIC, R3“, RSb, and R3c are the same. In some
embodiments, R18, R”, R”, R38, R3b, and R30 are deuterium, and R2, R4, R5, R6, R7, R8, R93,
Rm’, Rloa, Rmb, and R10c are deuterium or hydrogen. In yet another embodiment, R”, R”, R”,
R3“, R3“), and R3c is deuterium, and R2, R4, R5, R6, R7, R8, R9“, R917, R103, R‘“; and R‘°° are
hydrogen.
W0 2013f049726 PCT/USZ012/058127
In yet another embodiment, R4 is deuterium, and R”, R1“, R1“, R2, R33, R3”, R3“,
R5, R6, R7, R8, R98, R9b, R103, Rm, and R10c are deuterium or hydrogen. In other
embodiments, R4 is deuterium, and R1“, Rlb, R”, R2, R38, R3b, Rh, R5, R6, R7, R8, R93, Rm’,
R103, Rmb, and R100 are hydrogen.
In another embodiment, R5 is deuterium, and R13, R”, R”, R2, R38, R3b, R3“, R4,
R6, R7, R8, R98, Rgb, R108, wa, and ch are deuterium or hydrogen. In yet another
embodiment, R5 is deuterium, and R“, Rlb, R”, R2, 1132111311 R3“, R4, R6, R7, R8, R9“, R9b,
R108, Rmb, and R10c are hydrogen.
In another ment, at least one of R98 or R9b are the same. In other
embodiments, at least one of R9a or R9b are deuterium, and R13, R”, R”, R2, R3”, R3b, R3°, R4,
R5, R6, R7, R8, R10”, Km”, and R10C are deuterium or en. In some embodiments, at least
one of R9a and R9b are deuterium, and R”, R”, R”, R2, R33, R”, R3“, R4, R5, R6, R7, R8, R103,
R“, R10c are hydrogen.
In one embodiment, R6, R9a and R9b are the same. In some embodiments, R6,
R9“ and R9b are deuten'um, and R“: R“: R1“, R2, R3”, R3", R3“, R4, R5, R7, R8, R10“, R”, R‘““
are deuterium or hydrogen. In other embodiments, R6, R93 and Rgb are deuterium, and R”,
R”, R“, R2, R33, R3b, R“, R4, R5, R7, R8, R10“, wa, and 11”“ are hydrogen.
In some embodiments, R2, Rm“, Rm", and R100 are the same. In another
embodiment, R2, R10“, Rmb, and R10c are deuterium, and R1“, Rlb, R”, R“, R3b, R3°, R4, R5,
R6, R2 R8, R93, and R9b are deuterium or hydrogen. In yet another embodiment, R2, R10“,
Rmb, and R10: are deuterium, and R13, R“: R1“, R33, R3b, R3C, R“, R5, R6, R7, R8, R9“, and R9"
are hydrogen.
In some ments, R7 and at least two of R103, Rlob, or R10c are the same. In
another embodiment, R7 and at least two of Rwa, wa, or R10c are ium, and R1“, Rib,
R”, R2, R3“, R”, R3“, R4, R5, R6, R8, R9“, and R91) are ium or hydrogen. In yet another
embodiment, R7 and at least two of R103, Rmb, or R10c are deuterium, and R”, R"’, R”, R2,
R33, R”, R3“, R4, R5, R6, R8, R98, and Rgb are hydrogen.
In some embodiments, R”, R”, R”, R2, R3“, R3b, R30, and at least one of Rm“,
wa, or R10c are the same. In another embodiment, R”, R”, R”, R2, R3“, R3”, R32 and at least
one of Rloa, wa, or R100 are deuterium, and R4, R5, R6, R7, R8, R93, and R9‘) are deuterium or
en. In yet another embodiment, R”, R”, R”, R2, R3", RSb, R3“, and at least one of Rm",
R10”, or R100 are deuterium, and R4, R5, R6, R7, R8, Rga, and R9b are hydrogen.
In some embodiments, R13, R”, R1“, R3“, R3b, R3”, and R5 are the same. In
another embodiment, R13, Ru’, RIC, R33, R3b, R“, and R5 are deuterium, and R2, R4, R6, R7, R8,
PCT/U32012/058127
R9”, R9b, Rm“, Rmh, and R100 are deuterium or hydrogen. In yet another ment, R”, R”,
R“, R33, R”, R3°, and R5 are deuterium, and R2, R4, R6, R7, R8, Rg“, R91“, R103, Rm", and R1“
are en.
In other embodiments, R4 and R6 are the same. In another embodiment, R4 and
R6 are deuterium, and R“: R”, R”, R2, R33, R3b, R32 R5, R7, R8, R93, 119", R103, Rmb, and R”
are deuterium or hydrogen. In yet another embodiment, R4 and R6 are deuterium, and R13,
Riba RIC, R2, R3a, R3b, R3°, R5, R7, R8, R93, R91), R102!) R101), and Rioe are hydrogen.
In one embodiment, R2, R5, R9“, and R9b are the same. In some embodiments,
R2, R5, R93, and Rgb are deuterium, and R”, R”: R”, R33, R3b, R32 R4, R6, R7, R8, R103, Rm,
and Rloc are deuterium or hydrogen. In another embodiment, R2, R5, R93, and R91) are
deuterium, and R”, R“: R“, R3“, R3”, R3“, R4, R6, R7, R8, R103, R101: and R1°° are hydrogen.
In yet another embodiment, R”, R”, R1“, R2, R33, R”, R3“, R5, R6, R9“, R9211”,
Rmb, and R10c are the same. In some embodiments, Rla, Rlb, R1“, R2, R38, R3b, R3°, R5, R6,
R93, R9b, R103, Rm, and Rlo‘: are deuten'um, and R4, R7, and R8 are deuterium or hydrogen. In
other embodiments, R”, R“, R‘“, R2, R3“, R”, R3“, R5, R6, 119“, 11%, Rm“, R‘Ob, and R'“ is
deuterium, and R4, R7, and R8 are en.
In some embodiments, the les are as depicted in the compounds of the
disclosure including compounds in the tables below.
TableI
NH? 0"1 HN‘CQ NH? O’N\ HNx
\ \
N \ N \
l |
/N /N
H 033:0
N /i\
1—1 1-2
W0 2013I049726 2012/058127
SOziPr
Table II
NH2 O‘N\ HN‘ D
N D NH
\ NHz 0;\ 2 0'“
N \ HN+D
D HN+D \\
I D N \ D N \ D
/ N | D I
/N /N
Oj:0 o=s=o 03:0
11—] 11-2 11—3
WMNH —N ””2 041‘
HN~ HN‘
NI \ \
/N NW“”2°"‘\' I |
/N D /N
O=S=O
DOW 8:8 83
D 0
D D Y {0
11-4 II-S 11-6
NH2 0": HM\ NHZ 041' HN‘ NH2 o—Q HN\<D
\ \ \
N \ N \ N \ D
| l I
/N D /N D /N
0% o\\ 0%
0981/ 0481/ o’rSY
11—7 11-8 11—9
WO 49726 PCT/U82012/058127
NH2 04: HN\
11—12
l D D
/N D
o’zSY
11-15
NWINHz 04$
“N40
ms D
0 fit:
D D
11—18
NHZ O’r‘i HN\
I D
11—20 11—21
II-22
W0 2013l049726 PCT/U82012/058127
Compounds of this invention include those described generally , and are
further rated by the classes, subclasses, and species disclosed herein. As used herein, the
following definitions shall apply unless otherwise indicated. For es of this invention,
the chemical elements are fied in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general ples
of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University
Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5lh Ed., Ed.:
Smith, MB. and March, J., John Wiley & Sons, New York: 2001, the entire contents of
which are hereby incorporated by reference.
As described herein, a specified number range of atoms includes any r
therein. For e, a group having from 1—4 atoms could have 1, 2, 3, or 4 atoms.
As described herein, compounds of the invention may optionally be substituted
with one or more substituents, such as are illustrated generally herein, or as ified by
particular classes, subclasses, and species of the invention. It will be appreciated that the
phrase “optionally substituted” is used interchangeably with the phrase “substituted or
unsubstituted.” In general, the term ituted”, whether ed by the term “optionally”
or not, refers to the replacement of hydrogen ls in a given structure with the radical of a
specified substituent. Unless otherwise indicated, an optionally substituted group may have a
substituent at each substitutable position of the group, and when more than one position in
any given structure may be tuted with more than one substituent selected from a
specified group, the substituent may be either the same or different at every position.
Combinations of substituents envisioned by this invention are preferably those that result in
the formation of stable or chemically feasible nds.
Unless otherwise indicated, a substituent connected by a bond drawn from the
center of a ring means that the substituent can be bonded to any on in the ring. In
example i below, for instance, I1 can be bonded to any position on the pyridyl ring. For
bicyclic rings, a bond drawn through both rings indicates that the substituent can be bonded
from any position of the bicyclic ring. In example ii below, for instance, I1 can be bonded to
the 5—membered ring (on the nitrogen atom, for instance), and to the 6—membered ring.
/ /NW' 1
_I_(J1)5 )0—5
\ §.<NJI\/ (J
N H
i ii
PCT/U52012/058127
The term “stable”, as used herein, refers to compounds that are not substantially
d when ted to conditions to allow for their production, detection, recovery,
purification, and use for one or more of the purposes disclosed herein. In some embodiments,
a stable compound or chemically feasible compound is one that is not substantially altered
when kept at a temperature of 40°C or less, in the absence of re or other chemically
reactive conditions, for at least a week.
[0084} The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain
(i.e., unbranched), ed, or cyclic, substituted or unsubstituted hydrocarbon chain that is
completely saturated or that ns one or more units of unsaturation that has a single point
of ment to the rest of the molecule.
Unless otherwise specified, aliphatic groups contain 1—20 aliphatic carbon atoms.
In some embodiments, aliphatic groups contain l-lO aliphatic carbon atoms. In other
embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In still other
embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in yet other
embodiments aliphatic groups contain 1—4 aliphatic carbon atoms. Aliphatic groups may be
linear or branched, tuted or unsubstituted alkyl, alkenyl, or alkynyl groups. c
examples include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl,
n-butenyl, l, and tert-butyl. tic groups may also be cyclic, or have a combination
of linear or branched and cyclic groups. Examples of such types of aliphatic groups include,
but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, —CH2-
cyclopropyl, CH2CH2CH(CH3)—cyclohexyl.
The term “cycloaliphatic” (or “carbocycle” or “carbocyclyl”) refers to a
monocyclic C3-C8 hydrocarbon or ic C8-C12 hydrocarbon that is completely ted or
that contains one or more units of unsaturation, but which is not aromatic, that has a single
point of attachment to the rest of the molecule wherein any individual ring in said bicyclic
ring system has 3—7 members. Examples of liphatic groups include, but are not limited
to, cycloalkyl and cycloalkenyl . c examples include, but are not limited to,
exyl, cyclopropenyl, and cyclobutyl.
The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used herein means
non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring
members are an independently selected heteroatom. In some embodiments, the
“heterocycle”, “heterocyclyl”, or “heterocyclic” group has three to fourteen ring members in
which one or more ring members is a heteroatom independently selected from oxygen, sulfur,
nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.
Examples of heterocycles include, but are not limited to, 3—lH—benzimidazol—2—
one, 3-(1-alkyl)-benzimidazol-Z-one, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-
tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-
thiomorpholino, 3-thiomorpholino, 4—thiomorpholino, l—pyrrolidinyl, olidinyl, 3—
idinyl, l—tetrahydropiperazinyl, 2—tetrahydropiperazinyl, 3—tetrahydropiperazinyl, 1-
pipcridinyl, 2-piperidinyl, 3-pipcridinyl, l—pyrazolinyl, 3—pyrazolinyl, 4-pyrazolinyl, 5—
pyrazolinyl, l—piperidinyl, 2—piperidinyl, 3~piperidinyl, 4—piperidinyl, 2—thiazolidinyl, 3—
thiazolidinyl, 4—thiazolidinyl, l~imidazolidinyl, 2—imidazolidinyl, 4—imidazolidinyl, 5—
imidazolidinyl, indolinyl, tctrahydroquinolinyl, tctrahydroisoquinolinyl, benzothiolane,
benzodithiane, and 1,3 -dihydro-imidazol~2~one
Cyclic groups, (e.g. liphatic and heterocycles), can be linearly fused,
d, or spiroeyclic.
The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,
phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or
silicon; the quaternized form of any basic nitrogen or; a substitutable en of a
heterocyclic ring, for example N (as in 3,4—dihydro—2H—pyrrolyl), NH (as in pyrrolidinyl) or
NR+ (as in N-substituted idinyl)).
The term "unsaturated", as used herein, means that a moiety has one or more units
of unsaturation. As would be known by one of skill in the art, unsaturated groups can be
partially unsaturated or fully unsaturated. Examples of lly unsaturated groups include,
but are not limited to, butene, cyclohexene, and tetrahydropyridine. Fully unsaturated groups
can be aromatic, anti—aromatic, or non-aromatic. Examples of fully unsaturated groups
e, but are not limited to, phcnyl, cyclooctatctraene, pyridyl, thienyl, and l—
methylpyridin—2(1H)—one.
The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkyl group, as
previously defined, attached through an oxygen (“alkoxy”) or sulfi1r(“thioalkyl”) atom.
The terms “haloalkyl”, “haloalkenyl”, “haloaliphatic”, and “haloalkoxy” mean
alkyl, alkenyl or , as the case may be, substituted with one or more halogen atoms.
This term es perfluorinated alkyl groups, such as -CF3 and —CF2CF3.
The terms “halogen”, “halo”, and “hal” mcan F, Cl, Br, or I.
The term “aryl” used alone or as part of a larger moiety as in “aralkyl”,
“aralkoxy”, or “aryloxyalkyl”, refers to clic, bicyclic, and tricyclic ring systems
having a total of five to fourteen ring members, wherein at least one ring in the system is
W0 20131049726 PCT/U82012/058127
aromatic and wherein each ring in the system contains 3 to 7 ring members. The term “aryl”
may be used interchangeably with the term “aryl ring”.
The term “heteroaryl”, used alone or as part of a larger moiety as in
“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, ic, and lic ring
systems having a total of five to fourteen ring members, n at least one ring in the
system is aromatic, at least one ring in the system contains one or more heteroatoms, and
wherein each ring in the system contains 3 to 7 ring members. The term “heteroaryl” may be
used interchangeably with the term “heteroaryl ring” or the term “heteroaromatic”. Examples
of heteroaryl rings include, but are not limited to, 2-furanyl, 3—furanyl, N—imidazolyl, 2—
imidazolyl, 4—imidazolyl, 5—imidazolyl, benzimidazolyl, 3—isoxazolyl, 4—isoxazolyl, 5—
isoxazolyl, 2-oxazolyl, 4—oxazolyl, 5-oxazolyl, olyl, 2—pyrrolyl, 3-pyrrolyl, 2—pyridyl,
3—pyridyl, 4—pyridyl, 2-pyrimidinyl, 4—pyrimidinyl, 5—py1imidinyl, pyridazinyl (e.g., 3-
pyridazinyl), 2—thiazolyl, 4-thiazolyl, S—thiazolyl, tetrazolyl (e.g., 5—tetrazolyl), triazolyl (e. g.,
2—triazolyl and 5-triazolyl), 2—thienyl, 3—thienyl, benzofuiyl, benzothiophenyl, indolyl (e.g., 2—
l), pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1,2,3—oxadiazolyl, 1,2,5—oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,3—triazolyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5—thiadiazolyl,
l, pyrazinyl, 1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl),
and isoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl).
It shall be understood that the term “heteroaryl” includes certain types of
heteroaryl rings that exist in equilibrium between two different forms. More cally, for
example, species such hydropyridine and pyridinone (and likewise hydroxypyrimidine and
pyrimidinone) are meant to be encompassed within the definition of “heteroaryl.”
|\ |\
/N‘— NH
OH O
The term “protecting group” and “protective group” as used herein, are
interchangeable and refer to an agent used to temporarily block one or more desired
functional groups in a nd with multiple ve sites. In certain embodiments, a
protecting group has one or more, or preferably all, of the following characteristics: a) is
added selectively to a onal group in good yield to give a ted substrate that is b)
stable to reactions occurring at one or more of the other reactive sites; and c) is selectively
ble in good yield by reagents that do not attack the regenerated, deprotected onal
group. As would be understood by one skilled in the art, in some cases, the reagents do not
PCT/U82012/058127
attack other reactive groups in the nd. In other cases, the reagents may also react
with other reactive groups in the compound. Examples of protecting groups are detailed in
, T.W., Wuts, P. G in “Protective Groups in Organic Synthesis”, Third n, John
Wiley & Sons, New York: 1999 (and other editions of the book), the entire contents of
which are hereby incorporated by reference. The term “nitrogen protecting , as used
herein, refers to an agent used to temporarily block one or more desired nitrogen reactive
sites in a multifunctional compound. Preferred nitrogen protecting groups also possess the
characteristics exemplified for a protecting group above, and certain exemplary nitrogen
protecting groups are also detailed in Chapter 7 in Greene, T.W., Wuts, P. G in “Protective
Groups in Organic Synthesis”, Third Edition, John Wiley & Sons, New York: 1999, the
entire contents of which are hereby incorporated by reference.
In some embodiments, a methylene unit of an alkyl or aliphatic chain is optionally
ed with another atom or group. Examples of such atoms or groups include, but are not
d to, nitrogen, oxygen, sulfur, -C(O)-, -C(=N—CN)—, —C(=NR)-, —C(=NOR)—, —SO—, and
-SOz-. These atoms or groups can be combined to form larger groups. Examples of such
larger groups include, but are not limited to, -OC(O)—, —C(O)CO-, —COz-, -C(O)NR—, —C(=N—
CN), -NRCO-, ~NRC(O)O-, -SOzNR-, —NRSOz-, -NRC(O)NR-, -OC(O)NR-, and
-NRSOZNR-, wherein R is, for example, H or C1-5aliphatie. It should be understood that
these groups can be bonded to the methylene units of the aliphatic chain via single, double, or
triple bonds. An example of an al ement (nitrogen atom in this case) that is
bonded to the aliphatic chain via a double bond would be ~CH2CH=N—CH3. In some cases,
especially on the terminal end, an optional replacement can be bonded to the aliphatic group
via a triple bond. One example of this would be CHZCHZCHZCEN. It should be understood
that in this situation, the terminal nitrogen is not bonded to another atom.
] It should also be understood that, the term “methylene unit” can also refer to
ed or substituted methylene units. For example, in an isopropyl moiety [—CH(CH3)2], a
nitrogen atom (e.g. NR) replacing the first recited “methylene unit” would result in
dimethylamine [-N(CH3)2]. In instances such as these, one of skill in the art would
tand that the nitrogen atom will not have any additional atoms bonded to it, and the
“R” from “NR” would be absent in this case.
Unless otherwise ted, the al replacements form a chemically stable
compound. Optional replacements can occur both within the chain and/or at either end of the
chain; i.e. both at the point of attachment and/or also at the al end. Two optional
replacements can also be adjacent to each other within a chain so long as it results in a
W0 2013.1049726
chemically stable compound. For example, a C3 aliphatic can be ally replaced by 2
nitrogen atoms to form —C—N—=-N. The optional replacements can also completely replace all
of the carbon atoms in a chain. For example, a C3 aliphatic can be optionally replaced by
—NR-, —C(O)—, and -NR— to form —NRC(O)NR— (a urea).
] Unless otherwise indicated, if the replacement occurs at the terminal end, the
replacement atom is bound to a hydrogen atom on the terminal end. For example, if a
methylene unit of —CH2CH2CH3 were optionally replaced with -O-, the resulting compound
could be -OCH2CH3, -CH20CH3, or -CH2CH20H. It should be understood that ifthe
terminal atom does not contain any free valence electrons, then a hydrogen atom is not
required at the al end (e. g., -CH2CH2CH=O or ~CH2CH2CEN).
] Unless otherwise indicated, structures depicted herein are also meant to include all
isomeric (e.g., enantiomeric, reomeric, geometric, conformational, and rotational)
forms ofthe structure. For example, the R and S configurations for each tric center,
(Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this
invention. As would be understood to one d in the art, a substituent can freely rotate
around any rotatable bonds. For example, a substituent drawn as \ also
represents
Therefore, single chemical isomers as well as enantiomeric, diastereomeric,
geometric, conformational, and rotational mixtures of the t compounds are within the
scope of the invention.
Unless otherwise indicated, all tautomeric forms of the compounds of the
invention are within the scope of the invention.
In the compounds of this invention any atom not specifically designated as a
particular isotope is meant to represent any stable isotope of that atom. Unless otherwise
stated, when a position is designated cally as "H" or "hydrogen", the on is
understood to have hydrogen at its natural abundance isotopic composition. Also unless
otherwise stated, when a position is designated specifically as "D" or rium", the
position is understood to have deuterium at an abundance that is at least 3340 times greater
than the natural abundance of ium, which is 0.015% (i.e., at least 50.1% incorporation
of deuterium).
PCT/USZ012/058127
"D" and "d" both refer to deuterium.
Additionally, unless otherwise indicated, structures depicted herein are also meant
to include compounds that differ only in the presence of one or more isotopically enriched
atoms. For e, nds having the present structures except for the replacement of
hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C—enriched
carbon are within the scope of this invention. Such compounds are useful, for example, as
analytical tools or probes in ical assays.
Processes
] Processes and compounds described herein are useful for producing ATR
inhibitors that contain an aminopyrazine-isoxazole core. The general tic procedures
shown in schemes herein are useful for generating a wide array of chemical species which
can be used in the manufacture of pharmaceutical compounds.
SCHEME A
R1_O R1 0._
/ \ \ SteP 1 o / \ (Ra
SlepZ Rl-O ,R3
-|_—jt t N N
‘_» H _—>
R2_O R2- ‘ :/
Protection R -O2 IIDG
Reductuve
J1 amination J1 J1
1 2 g
N 0’” ,R
/ (\E‘G‘
Step3 HO-N ,R3 Step4 \\ Step5
—* \ /l_)wI? ———> N \ "'— --—-—>
Oxime PG lsoxazole J1 Suzuki
formation J1 formation "\(N (when R4 is Br)
4 (1 or 2 steps) R4
Deprotection
NH2 o—N 3 NH O’N 3 NH 0~N 3
\\ / ”NR 2
Step6 \\ / ”N’R 2
Step7 \\ N’R
1:) H —-———> hi \ H ————-——> H
free base 1:) N' \ NI \
RfN KrN saItforma Iont'
J1 J1 YN . acid ion J1
R4 R, R,
I [—A 1-3
Step 1
The compound of formula I can be made according to the steps outlined in
Scheme A. Step 1 depicts the use of a y available aldehyde/ketal as a starting point for
the preparation of compounds of formula 1, LA, and LB. Reductive amination between
compound _1_ and a suitable primary amine, under conditions known to those skilled in the art
leads to compound _2_ where a benzylamine motif has been installed. For example, imines can
be formed by combining an amine and an aldehyde in a suitable solvent, such as
dichloromethane (DCM), dichloroethane (DCE), an alcoholic solvent (e. g., methanol,
ethanol), or a nonprotic solvent (e.g., dioxane or tetrahydrofuran (THF)). These imines can
then be reduced by known reducing agents including, but not limited to, NaBH4, N,
and NaBH(OAc)3 (sfl JOC 1996, 3849). In some embodiments, 1.05 equivalents of amine is
combined with 1 lent of aldehyde in methanol. In other embodiments, 1.2 lents
of amine is combined with 1 equivalent of aldehyde in methanol. This step is then followed
by reduction with 0.6 to 1.4 (such as 1.2) equivalents ofNaBH4. In some cases, if an amine
salt is used, base (e.g., Et3N or ropylethylamine) can also be added.
Step 2 depicts the protection of the benzylamine _1_ ed above, using a
carbamate—based protecting group, under suitable protection conditions known to those
d in the art. Various protecting groups, such as Cbz and Boo, can be used. Protection
ions include, but are not limited to the following:
a) R-OCOCl, a suitable tertiary amine base, and a suitable solvent; wherein R is
C1-6alkyl optionally substituted with phenyl;
b) R(C02)OR’, a suitable t, and optionally a catalytic amount of base, wherein
R is and R’ are each independently C1_5alkyl optionally substituted with phenyl;
c) [RO(C=O)]20, a suitable base, and a suitable solvent.
Examples of suitable bases include, but are not limited to, EN,
ropylamine, and pyridine. Examples of suitable solvents include nated solvents
(e.g., dichloromethane (DCM), dichloroethane (DCE), CHzClz, and chloroform), ethers (e. g.,
, 2-MeTHF, and dioxane), aromatic hydrocarbons (e.g., toluene, xylenes) and other
aprotic solvents.
In some embodiments, protection can be done by reacting the amine with
0 and Et3N in DCM. In some embodiments, 1.02 equivalents of (Boc)20 and 1.02
equivalents ofEth 1.02 are used. . In another embodiment, protection can be done by
reacting the benzylamine with (Boc)20 in 2—MeTHF. In some embodiments, 1.05 equivalents
of (Boc)20 are used.
Step 3
Step 3 shows how the ketal functional group in s is then converted into the
oxime fl in a single step. This direct conversion from ketal to oxime is not extensively
bed in the literature and it will be appreciated that this step could also be conducted in a
two-step sequence, transiting through the aldehyde after deprotection of the ketal using
methodologies known to those skilled in the art.
Oxime formation conditions comprise mixing together hydroxylamine, acid,
optionally a dehydrating agent, and an alcoholic solvent. In some embodiments, the acid is a
catalytic amount. In some embodiments, the acid is pTSA or HCI, the dehydrating agent is
molecular sieves or dimethoxyacetonc, and the alcoholic solvent is methanol or ethanol. In
some embodiments, the hydroxylamine hydrochloride is used in which case no additional
acid is required. In other embodiments, the desired product is isolated via a biphasic work up
and optionally precipitation or llization. If a biphasic work up is used, a dehydrating
agent is not .
] In another embodiment, the oxime formation conditions comprise of mixing
together ylamine, an acid, an organic solvent and water. Examples of suitable organic
solvents include chlorinated solvents (e.g., dichloromethane (DCM), roethane (DCE),
CH2C12, and form), ethers (e.g., THF, 2—MeTHF and dioxane), aromatic hydrocarbons
(e.g., toluene, xylenes) and other aprotic solvents. In some embodiments, 1.5 equivalents of
ylamine hloride are used, the organic solvent is 2-MeTHF and the water is
buffered with NaZSO4. In another embodiment, 1.2 equivalents of hydroxylamine
hloride are used, the organic solvent is THF.
In some embodiments, suitable deprotection conditions comprise adding
catalytic amounts of para—toluenesulfonic acid (pTSA), acetone, and water; and then forming
the oxime using conditions known to one skilled in the art. In other embodiments, a single
step sequence is used. In some embodiments, the single step sequence ses adding
NHZOHHCI and a mixture ofTHF and water. In some embodiments, 1 equivalent of the
compound of a 3 is combined with a 1.1 equivalents ofNHZOHHCI in a 10:1 v/V
mixture of THF/water.
Step 4
Step 4 illustrates how the oxime fl is then transformed and engaged in a [3+2] cycloaddition
to for the ole Q. This transformation can be conducted in one pot but requires two
distinct steps. The first step is an oxidation of the oxime functional group into a nitrone, or a
similar intermediate with the same degree of oxidation, for example a chlorooxime. This
reactive species then reacts with an alkyne in a [3+2] cycloaddition to form the ole
adduct.
W0 2013f049726 PCT/U32012/058127
In some embodiments, the suitable ole—formation ions ts of two
steps, the first step sing reacting the compound of formula 4 under suitable
chlorooxime formation conditions to form a chlorooxime intermediate; the second step
comprising ng the chlorooxime intermediate with acetylene under suitable
cycloaddition conditions to form a compound of formula 5.
] In some embodiments, the chlorooxime formation ions are selected from
a) N—chlorosuccinimide and suitable solvent;
b) potassium peroxymonosulfate, HCl, and dioxane; and
c) Sodium hypochlorite and a suitable solvent
Examples of suitable solvents include, but are not limited to, nonprotic solvents
(e.g., DCM, DCE, THF, 2—MeTHF, MTBE and e), aromatic hydrocarbons (e.g.
toluene, xylenes), and alkyl acetates (e. g., isopropyl acetate, ethyl acetate).
Isolation of the product can be achieved by adding an antisolvent to a solution of a
compound of formula 5. Examples of suitable solvents for isolating the chlorooxime
intermediate include mixtures of suitable solvents (EtOAc, IPAC) with arbons (e.g.,
hexanes, heptane, cyclohexane), or aromatic hydrocarbons (e.g., toluene, xylenes). In some
embodiments, heptane is added to a solution of chlorooxime in IPAC.
Suitable cycloaddition conditions consist of combining the chlorooxime with
ene with a suitable base and a suitable solvent. Suitable solvents include protic
solvents, aproptic solvents, polar solvents, and nonpolar solvents. Examples of suitable
solvent include, but are not limited to, acetonitrile, tetrahydrofuran, 2—methy1tetrahydrofuran,
MTBE, EtOAc, c, DCM, toluene, DMF, and ol. Suitable bases include, but are
not limited to, pyridine, DIEA, TEA, t—BuONa, and K2C03. In some embodiments, suitable
cycloaddition conditions comprise adding 1.0 equivalents of oxime, 1.0 lents of
acetylene, 1.1 equivalents of Et3N in DCM.
Isolation of the product can be achieved by adding an lvent to a solution of a
compound of formula 5. Examples of suitable solvents for isolating the chlorooxime include
mixtures of suitable solvents , IPAC) with hydrocarbons (e.g., hexanes, heptane,
cyclohexane), or aromatic hydrocarbons (e.g., toluene, xylenes). In some embodiments,
heptane is added to a solution of chlorooxime in IPAC.
Step 5
Step 5 depicts the final step(s) of the preparation of compounds of formula I.
When the R4 group is bromo, intermediate i can be subjected to a Suzuki cross—coupling
W0 2013/049726 PCT/U82012/058127
with boronic acid or esters, under conditions known to those d in the art, to form
compounds Where R4 an aryl, heteroaryl or alternative moieties resulting from the metal-
assisted ng reaction. When intermediate Q is ly functionalised, a deprotection step
can be carried out to remove the protecting groups and generate the compounds of formula I.
Metal assisted coupling reactions are known in the art (se_e e.g., oc. Res.
Dev. 2010, 30-47). In some embodiments, suitable coupling conditions comprise adding 0.1
equivalents of Pd[P(tBu)3]2; 1 equivalent of c acid or ester; and 2 equivalents of
sodium ate in a 2:1 ratio v/v of acetonitrile/water at 60—70°C. In other embodiments,
suitable coupling conditions se adding 0010—0005 equivalents Pd(dtbpt)Clg, 1
equivalent of boronic acid or ester, and 2 equivalents of potassium carbonate in a 7:2 v/v of
toluene and water at 70 °C
The final product can treated with a metal scavenger (silica gel, fiinctionalized
resins, charcoal) (sgge.g., Org. Proc. Res. Dev. 2005, 198-205). In some embodiments, the
solution of the product is treated with Biotage MP—TMT resin.
The product can also be isolated by crystallization from an lic solvent
(e. g. methanol, ethanol, isopropanol). In some embodiments the solvent is ethanol. In other
embodiments the solvent is isopropanol.
Deprotection of B00 groups is known in the art (see eg. Protecting Groups in
Organic Synthesis, Greene and Wuts). In some embodiments, suitable deprotection
conditions are hydrochloric acid in acetone at 35—45 °C. In other embodiments, suitable
deprotection conditions are TFA in DCM.
Step 6
] Step 6 illustrates how compounds of formula I are converted to compounds of
a I—A using a base under suitable conditions known to those skilled in the art. In some
embodiments, isolation of the free-base form of compounds of formula I may be achieved by
adding suitable base, such as NaOH to an alcoholic acidic solution of compounds of formula
Ito precipitate the t.
$12.2
Step 7 illustrates how compounds of a I—A are ted to compounds
of formula I-B using an acid under syuitable conditions known to those skilled in the art.
In some embodiments suitable conditions involve adding aqueous HCl, to a
sion of compounds of formula LA in acetone at 35 °C then heating at 50 °C.
PCTIU82012/058127
SCHEME B: Formation of dl—boronate
x W
x O\,0
1) Base te
2) 020 Formation
—————> ——————>
O=S=O
Scheme B shows a general synthetic method for the preparation of dl—boronate
intermediates. A suitable 1-halo-(isopropylsulfonyl)benzene is treated with a base such as,
but not d to NaH, LiHMDS or KHMDS followed by quenching of the anion with
deuterium source such as D20. The halogen is then transformed into a suitable boronate
derivative via, for example, metal mediated cross—coupling catalyzed by, for instance,
3)2 or Pd(dppt)Clz-DCM.
SCHEME C: Formation of onate
x W
x O\,0
1) Base Boronate
2) DECX Formation
__—_.).
023:0
o=s=o
| DWD 0:3:0
D D
D D 3W3
D D
] Scheme C shows a general synthetic method for the preparation of d6-boronate
intermediates. A suitable 1-halo-(methylsulfonyl)benzene is d with a base such as, but
not limited to NaH, LiHMDS or KHMDS followed by quenching of the anion with deuterium
source such as D3CI. This reaction is repeated until the desired amount of deuterium has been
incorporated into the molecule. The halogen is then transformed into a suitable boronate
derivative via, for example, metal mediated cross-coupling catalyzed by, for instance,
Pd(tBu3)2 or Pd(dppf)C12-DCM.
PCT/U52012/058127
SCHEME D: Formation of d7-boronate
Br Br
Br W
< 1 © © 0~ ,0
Boronate
Alkylation 3 m O=S=O ion
SH “ ’ [3WD 0WD “————> 0-3—0
D D D D D D
D D D D D
D D D
D D
] Scheme D shows a general synthetic method for the preparation of d7—boronate
intermediates. 4—Bromobenzenethiol is treated with a base such as, but not limited to NaH,
LiHMDS or KHMDS ed by quenching of the anion with deuterium source such as
1,1,1,2,3,3,3—heptadeuterio~2-iodo-propane. The sulfide is then oxidized to the ponding
e using, for example, mCPBA or Oxone. The n is then transformed into a
suitable boronate derivative via, for example, metal mediated cross-coupling catalyzed by, for
instance, Pd(‘Bu3)2 or Pd(dppf)Clg-DCM.
SCHEME E: Formation of aryl ring deuterated boronate
Br Br W W
Br 0e9,0 0‘5,0
/ ' /
/ ' '
' DI +x +x Boronate
TX splacernent \ on \ Formation i}X Dauterogenaflon @-
\ __—'> D
l I
\ \
s o=s=o
l; 323“ ”I, til,
Br O‘BD O‘B,O
©X/ __-_ Emma.” / Deuterogenation /
Formation +X ————> +5
\ \
0:0 o=s=o o=s=o
D D
Scheme B shows a general synthetic method for the preparation of boronate
intermediates where the aryl ring is substituted with a deuterium. A suitable l-iodo—4—bromo—
aryl derivative is treated with a substituted thiol such as propane—Z-thiol under metal
catalyzed coupling conditions using a catalyst such as CuI. The sulfide is then oxidized to the
corresponding e using, for example, mCPBA or Oxone. The bromide is then
transformed into a suitable boronate derivative via, for example, metal mediated cross-
eoupling catalyzed by, for instance, 3)2 or Pd(dppf)C12-DCM. The remaining
substituent is then converted into deuterium by, for instance, metal catalyzed halogen—
deuterium exchange using a suitbale metal catalyst, such as Pd on C under an atmposhere of
deuterium gas. In addition, the l—bromo—(isopropylsulfonyl)benzene can be treated with a
base such as, but not limited to NaH, LiHMDS or KHMDS followed by quenching of the
anion with deuterium source such as D20. The bromide is then transformed into a suitable
boronate derivative via, for example, metal mediated cross-coupling catalyzed by, for
instance, Pd(‘Bu3)2 or Pd(dppf)Clz-DCM. The remaining substituent is then converted into
deuterium by, for instance, metal catalyzed n-deuterium exchange using a le
metal catalyst, such as Pd on C under an atmposhere of deuterium gas.
SCHEME F: Formation of aryl ring deuterated boronate
Br Br
B! W
0‘ ,0
. /-:-X /—;X 0‘3’0
\TX \ \
~ I
Alkylahon Oxldation Boronate ~ /
_ _ _'x Deuterogenation
Formation \
SH [5&0 0:93;?) __:_D
O=S=O
DDDDD DDDDD 0=5=0
D D
D D D
D D 3 D g
D D
Scheme F shows r general tic method for the preparation of
boronate ediates where the aryl ring is substituted with a deuterium. A substituted 4-
bromobenzenethiol is treated with a base such as, but not limited to NaH, LiHMDS or
KHMDS followed by quenching of the anion with deuterium source such as 1,] ,1,2,3,3,3—
heptadeuterio-2—i0donpropane. The sulfide is then oxidized to the corresponding e
using, for example, mCPBA or Oxone. The halogen is then transformed into a suitable
boronatc derivative via, for example, metal mediated cross—coupling catalyzed by, for
ce, Pd(tBu3)z or Pd(dppf)Clz'DCM. The remaining substituent is then converted into
deuterium by, for instance, metal catalyzed halogen-deuterium exchange using a suitbale
metal catalyst, such as Pd on C under an atmposhere of deuterium gas.
SCHEME G: Formation of aryl ring deuterated boronate
Br Br
Br /
/ / TX
/ . _._X .1.X 1)Base \
-—x_ Alkylatlon Bumnala
I Oxidatlon l
l —_).. \
\ _____.) \ __).JC_X_>2 D Formation
O=S=O —->
S 0=S=O
SH D D
[ l
D D
D D
0‘ ,O 0» ,0
/ /
—'X Deutamganation +0
\ \
O=S=O O=S=O
D D
D D DownD
D D
W0 2013f049726 PCT/U52012/058127
Scheme G shows another general synthetic method for the preparation of boronate
intermediates where the aryl ring is substituted with a deuterium. A tuted 4-
bromobenzenethiol is treated with a base such as, but not limited to NaH, LiHMDS or
KHMDS ed by quenching of the anion with for instance Mel. The sulfide is then
oxidized to the corresponding sulfone using, for example, mCPBA or Oxone. The e is
treated with a base such as, but not d to NaH, LiHMDS or KHMDS followed by
ing of the anion with deuterium source such as D3CI. This on is repeated until
the desired amount of deuterium has been incorporated into the molecule. The halogen is then
transformed into a suitable boronate derivative via, for example, metal mediated cross—
coupling catalyzed by, for instance, Pd(tBu3)2 or f)C12-DCM. The remaining
substituent is then converted into deuterium by, for instance, metal catalyzed n-
deuterium exchange using a suitbale metal st, such as Pd on C under an atmposhere of
deuterium gas.
SCHEME H: Formation of aryl ring deuterated oxime intermediates
3, L
0/ / O
I 3' / I
x] 1 o\ ernination Hydrolysis 7‘ 0\ Protection /\0 /
’ I
O\M -‘—‘>
7‘ x |
‘/ 0
O X 0 X\ \
' O
k0 to L
Hoductwe. ~
/\o / mm“ Aom M /\O Amination
I __._._>
7\ o x o l
\ D/ \ \/
D/ o
o 0
300 O 0
/\o /\0 \ 0Y0 Oxlme Formatlon \ Y
\ Protection
I H ————————> I l
I/ N R10 // N Rm 0// N\'<H‘°11
D D
RD- away?" RD- gnaw?" Figa Rwfi1
Scheme H shows a general synthetic method for the preparation of oxime
intermediates where the aryl ring is substituted with a deuterium. The methyl group of an
appropriately substituted methyl ALmethylbenzoate derivative can be converted into the
corresponding dibromide under conditions such as AIBN catalyzed bromination with NBS.
This di-bromide is then hydrolysed to the corresponding aldehyde, for instance using AgN03
in acetone/water. tion of the aldehyde as a suitable acetal, for instance the diethyl acetal
and subsequent conversion of the remaining substituent into deuterium by, for instance, metal
catalyzed halogen-deuterium exchange using a suitable metal catalyst, such as Pd on C under
an atmosphere of deuterium gas gives the deuterated ester intermediate. The ester
W0 20131049726 PCT/U52012/058127
functionality can be reduced using reagents such as LiAlH4, NaBH4, NaBD4 or LiAlD4 to
give corresponding aldehyde. This can be reacted under reductive amination ions using
a suitable amine, such as methylamine or d3-methylamine using a reducing agent such as
NaBH4 or NaBD4 to give the corresponding amine tive. This can be protected with, for
instance a Boc group and the acetal converted into the oxime using, for instance,
hydroxylamine hydrochloride in THF/water.
SCHEME 1: Formation of aryl ring deuterated oxime intermediates
\ 0/ / ‘o
I 3’ / I
X] I o\ Bromination I Hydrolysis 7~ 0\ Protection /
0\ *—’ “'_’ \0
7» x '
O X ~/
O X‘ o\
0 \O \O
Deuterogenation~ \0 / - \o \ \
Amide FormationA Reduction- 0 \ BOG
———-—————> I —-———-—-——> I —-—-—-—> I _
7~ 0\ // NH; D// NHZm D D
0 0 R9“ F19"
\O \l/ \O \{/ H0~IN
\o O
\ 0Y0 Alkytation \o o 0 O
\ Oxime Formation \
I I I
// NH D// N R10
D D// N¥R1°
Rea R» R" " R!» R” at?"
Scheme 1 shows another general synthetic method for the preparation of oxime
ediates Where the aryl ring is substituted with a deuterium. The methyl group of an
appropriately substituted methyl 4—methylbenzoate derivative can be converted into the
corresponding dibromide under conditions such as AIBN zed bromination with NBS.
This di—bromide is then hydrolysed to the corresponding aldehyde, for instance using AgNO3
in acetone/water. Protection of the aldehyde as a suitable acetal, for instance the dimethyl
acetal and subsequent conversion of the remaining subtituent into deuterium by, for instance,
metal catalyzed halogen-deuterium exchange using a suitable metal catalyst, such as Pd on C
under an here of deuterium gas gives the deuterated ester intermediate. The ester
onality can be converted into the ponding primary amide under standard
conditions, such as g with a solution of ammonia in methanol. The amide can be
reduced to the corresponding amine using ts not d to LiAlH4 or LiA1D4. This can
be protected with, for instance a Bee group. The carbamate NH can be alkylated under basic
conditions using for instance NaH, LiHMDS or KHMDS followed by quenching of the anion
with deuterium source such Mel or D3CI. The acetal can be converted into the oxime using,
for instance, hydroxylamine hydrochloride in THF/water.
SCHEME J: Formation of aryl ring deuterated oxime intermediates
/ 0’ / \O
x/‘l o\ Byomlnatlnn 5’
I Hydrolysis 7,] o\ Protectlon \0 /
o X 0
\o \ \
o o
/ \
Deuterogenalion \0 I Amide Fonnafion \0 I Reduction \0 \
I 22?:3:
7‘ O\ ——)- // NH; ——————->~ // NH: ————>
D D D
O 0 R8- Rab
\o \O \i/ HoaN \I/
\0 \ O O
I \ Oxlme Formation \
H Satcect‘mn \o
I 10 I
/ "\KR ———> I
// N a") 1/ N am
D u D R9951?“ D
39' Rwfi‘ Fl“ R‘" Roam?"
Scheme J shows another general synthetic method for the preparation of oxime
intermediates where the aryl ring is tuted with a ium. The methyl group of an
appropriately substituted methyl y1benzoate derivative can be converted into the
corresponding dibromide under conditions such as AIBN catalyzed bromination with NBS.
This di—bromide is then hydrolysed to the corresponding aldehyde, for instance using AgN03
in acetone/water. Protection of the aldehyde as a suitable acetal, for instance the yl
acetal and subsequent conversion of the remaining substituent into deuterium by, for instance,
metal catalyzed n-deuterium ge using a suitable metal catalyst, such as Pd on C
under an atmposhere of deuterium gas gives the ated ester intermediate. The ester
functionality can be converted into the corresponding primary amide under standard
conditions, such as heating with a solution of ammonia in methanol. The amide can be
reduced to the corresponding amine using reagents not limited to LiAlH4 or LiAlD4. This can
be reacted under reductive amination conditions using a suitable amine, such as amine,
d3 -methylamine, formaldehyde or d2-formaldehyde using a reducing agent such as NaBH4 or
NaBD4 to give the corresponding amine tive. This can be protected with, for instance a
Boc group. The acetal can be converted into the oxime using, for instance, hydroxylamine
hydrochloride in THF/water.
PCT/U82012/058127
SCHEME K: Formation of aryl ring ated oxime intermediates
an R9‘1
/ /
I o
I /
x o Bmmination Br
\ ———-—> I Hydrolysis Deuterogenation 7‘ 0
X 7‘ o\ \
0 X 0
o \4/
$333: \0 ° 0
Protection \
l Alkylafion
/R5' R9"2 D// NH \an
R93 R9” /R9‘ Rim?"
Reduction Oxldalion II \ DY Oxlme Formatlon
R9131?KR“) / / N R10 D/ / N H10
11 D K \K 11
R9“ Rah R15" Fl“ R“ R‘
Scheme K shows another general synthetic method for the preparation of oxime
intermediates where the aryl ring is substituted with a deuterium. The methyl group of an
appropriately substituted methyl 4-methylbenzoate derivative can be converted into the
ponding ide under ions such as AIBN catalyzed bromination with NBS.
This di—bromide is then hydrolysed to the corresponding aldehyde, for instance using AgN03
in acetone/water. Protection of the aldehyde as a suitable acetal, for instance the dimethyl
acetal and subsequent conversion of the remaining substituent into deuterium by, for instance,
metal catalyzed halogen-deuterium exchange using a suitable metal catalyst, such as Pd on C
under an atmosphere of deuterium gas gives the deuterated ester intermediate. This can be
reacted under ive amination conditions using a suitable amine, such as um
hydroxide using a reducing agent such as NaBH4 or NaBD4 to give the corresponding amine
tive. This can be protected with, for instance a Boc group and the carbamate NH
alkylated under basic conditions using for instance NaH, LiHMDS or KHMDS followed by
quenching of the anion with ium source such Mei or D3CI. The ester can be reduced to
the corresponding alcohol using a suitable reducing agent such as LiBH4 or NaBH4. The
alcohol can be ed to the aldehyde using regeants such as MnOz or Dess—Martin
periodane. The acetal can be converted into the oxime using, for instance, aqueous
hydroxylamine.
2012/058127
SCHEME L: ion of ated oxime intermediates
r Lo Lo \4/
O Reductive BOC
Aminafion /\O Protection /\O
/\ H 0Y0
o ——> ——>
N mo N R10
0 11
Han REDS?" Ran R9:Rl<1
Hot'N \i/
0 O
Oxime Formation
N R10
RSI: nab}:
Scheme L shows a general synthetic method for the preparation of deuterated
oxime intermediates. 4-(diethoxymethyl)benzaldehyde can be reacted under reductive
amination conditions using a suitable amine, such as methylamine or d3 —methy1amine using a
ng agent such as NaBH4 or NaBD4 to give the corresponding amine derivative. This
can be protected with, for instance a Boc group and the acetal converted into the oxime using,
for instance, hydroxylamine hydrochloride in THF/water.
SCHEME M: Formation of deuterated oxime intermediates
\0 \O \O \O \l/
\0 Amide Formation \0 Reduction \0 ggiiacllon \0
.—-—> ——* 0Y0
0\ NH, NH: ——> NH
0 o R9I Fl” R" Rob
\0 \P Hos“ \k
Alkylation \o 0Y0 Oxima Formation
’ ’ 0Y0
NXR“) NXRW
R“ R“b R1?“ R“ nwmf‘"
Scheme M shows another general synthetic method for the preparation of
deuterated oxime intermediates. The ester functionality of methyl 4—
(dimethoxymethyl)benzoate can be converted into the corresponding primary amide under
standard conditions, such as heating with a solution of ammonia in ol. The amide can
be reduced to the corresponding amine using reagents not limited to LiAlH4 or LiAlD4. This
can be protected with, for instance a Boc group. The carbamate NH can be ted under
basic conditions using for instance NaH, LiHMDS or KHMDS followed by quenching of the
anion with deuterium source such Mel or D3CI. The acetal can be converted into the oxime
using, for instance, ylamine hydrochloride in THF/water.
WO 49726 PCT/USZ012/058127
SCHEME N: Formation of ated oxime intermediates
\ \
o o \
\0 Amide Formation \0 Reduction \0 WEEK:
0\ ——‘> NH2 ——"‘> NH2 ————>
o o R" fish
O \O \i/ HO‘N \1/
\ I
H Eggctlon \0 Oxime Formation
NXRw
0Y0 0Y0
-——-~——> N R10 N R10
Fififl HWRi \l< \|< 11
R“ R3" R15“ R9: 3% R:
Scheme N shows another general synthetic method for the preparation of
deuterated oxime intermediates. The ester functionality of methyl 4-
(dimethoxymethyl)benzoate can be converted into the corresponding primary amide under
standard conditions, such as heating with a solution of ammonia in methanol. The amide can
be d to the corresponding amine using reagents not limited to LiAlH4 or LiAlD4. This
can be reacted under reductive amination conditions using a suitable amine, such as
methylaminc, d3-methylaminc, dehyde or dZ—formaldehyde using a reducing agent
such as NaBH4 or NaBD4 to give the ponding amine derivative. This can be protected
with, for instance a Boc group. The acetal can be converted into the oxime using, for
instance, hydroxylamine hydrochloride in THF/water.
SCHEME 0: Formation of deuterated oxime ediates
0 \l/ o 4/
\0 Protection \0 0Y0 Alkylatlon \0
_..~._> ____._.> 0Y0
"“2 NH
F19“ R9” NKRW
R9: Rab R98 RWR1E“
\l/ (I) \l/ HoxN \l/
Reduction HO 0Y0 Oxidation 0Y0 Oxime Formation
"*9 a a 0Y0
N n10 N n10 N R‘”
K5" w< .1 x
R9. Rab R1 R93 R9°n1 Rea Room?“
Scheme 0 shows another general synthetic method for the preparation of
deuterated oxime intermediates. A 4-substituted benzylmine can be protected with, for
instance a Boc group. The carbamate NH can be alkylated under basic conditions using for
ce NaH, LiHMDS or KHMDS followed by quenching of the anion with deuterium
source such Mel or D3CI. The ester can be reduced to the corresponding alcohol using a
suitable reducing agent such as LiBI-I4 or NaBH4. The l can be oxidized to the
aldehyde using regeants such as MnOz or Dess-Martin periodane. The acetal can be
ted into the oxime using, for instance, aqueous hydroxylamine.
PCT/U82012/058127
SCHEME P: ion of isoxazole derrivatives
(306)2N
NH2 NH, TMS (30cm ms
/ /
Br / / H5
N \ N \ BOG N \
YN1 _E§._,Son ashlra KKNI Protection I 2 ms Rama]1;Suzukl
.__._.___> /N —.——.>
Br Br Br O=S=O
R1u Rae
HI I: 92 R32:
R1: R3'
[3+2] cycloaddlton
HQIN \i/ HO‘IN \i/
0Y0 nation Cl 03/0
N am N aw
“7 ”K511 "7
R3 R R‘ RB Re” R”*5“R1
0 NH; o—N HN
0 \ Rn
(BOChN 04.: ‘4\N‘6H;:1 NWR12 \ I R9. R9”
N \ RI2 /N R7
I 9" no
/N R7 R
R3 R5
Deprotectiun
R5 ' "
R. o=s=o
9‘“ “3°
o=s=o
R“ R2 R3”
Rt: Ric
R1: R33
nib R2 fish
Rm FF“
Scheme P shows a general synthetic method for the preparation of ated
ne-isoxazole derrivatives. 3,5-Dibromopyrazin-2—amine is converted into the
corresponding silyl-protected alkyne under standard Sonagashira conditions utilizing, for
example, Pd(PPh3)4 and Cul as catalysts. The ne NH; can then be protected as, for
example the di~Boc derivative. Coupling of the pyrazine bromide with a boronate, for
instance those outlined in Schemes l to 6 above, under standard Suzuki cross—coupling
conditions followed by removal of the silyl protecting group give the desired alkyne
intermediate. Oximes, such as those outline in Schems 7 to 14 above, can be converted into
the corresponding chlorooximcs using, for instance, NCS. The alkyne and oxime
intermediates can o a [3+2] cycloaaditon to give corresponding isoxazole under
standard conditions, for instance by the addition of Et3N. The Boc protecting groups can be
removed under acicid conditions such as TFA in DCM or HCl in MeOH/DCM to give the
deuterated pyrazine isoxale derrivatives.
W0 2013I049726
SCHEME Q: Formation of deuterated isoxazole derrivatives
O 0
NH2 o—N F30 10
\\ HN‘éan" FsckNH O"!
N \ ‘(N‘éfifiu
I Rae N \ File
/N R7 R9”
l R“
n8 /N W R9b
Protection R5 Haloganation
——-—-———> _—>
O=S=O
R13 Rae
R1!) R2 R30
R” R3“ an R2 R38:
. W
R5 Deprotection Deuterogenalion
—-——)> -—--~———>-
o=s=o
O=S=O
R1: Rae
Rib H2 R35
Rte R33
Scheme Q shows a general tic method for the preparation of deutcrated
isoxazole derrivatives. The pyrazine NH; and benzylamineamine NH can be protected under
standard conditions using trifluoroacetic anhydride. Halogenation of the isoxazole ring with,
for example NIS followed by removal of the trifluoroacetate protecting group under basic
ions provides the desired halogenated interemdiates. The halogen can then converted
into deuterium by, for instance, metal catalyzed halogen—deuterium exchange using a suitbale
metal st, such as Pd on C under an atmposhere of deuterium gas.
Abbreviations
The ing iations are used:
ATP adenosine triphosphate
Boc tert-butyl carbamate
Cbz Carboxybenzyl
DCM dichloromethane
PCT/USZ012/058127
DMSO dimethyl sulfoxide
Et3N triethylamine
F 2-methyltetrahydrofuran
NMM N-Methyl morpholine
DMAP 4-Dimethylaminopyridine
TMS Trimethylsilyl
MTBE methyl tertbutyl ether
EtOAc ethyl e
i—PrOAc isopropyl acetate
IPAC isopropyl acetate
DMF dimethylformamide
DIEA diisopropylethylamine
TEA triethylamine
t—BuONa sodium teitbutoxide
K2C03 ium carbonate
PG Protecting group
pTSA para-toluenesulfonic acid
TBAF Tetra—n-butylammonium fluoride
1HNMR proton nuclear magnetic resonance
HPLC high performance liquid chromatography
LCMS liquid chromatography—mass spectrometry
TLC thin layer chromatography
Rt retention time
SCHEMES AND EXAMPLES
The compounds of the disclosure may be prepared in light of the cation using
steps generally known to those of ordinary skill in the art. Those compounds may be
analyzed by known methods, ing but not limited to LCMS (liquid chromatography
mass ometry) and NMR (nuclear magnetic resonance). The following generic schemes
and examples illustrate how to prepare the compounds of the present disclosure. The
examples are for the e of illustration only and are not to be construed as limiting the
scope of the invention in any way. H—NMR spectra were recorded at 400 MHz using a
Bruker DPX 400 instrument. Mass spec. samples were analyzed on a MicroMass Quattro
Micro mass spectrometer ed in single MS mode with electrospray ionization.
1Compound I-lz
£2 J
o\ HN NU
Method 1 Method 2 Method 3 JL J<
’ -—-————> N 0
m cm A o/\ Ho \«GA
o a, om 61
J< 0
i J< JOL 5;
N O >L0 -N Q 0 N 0°
Method4 Methods \ N~< 22 ..___, N
H0, \ -—-—————> \
CI I
0 /N
J< °
>qu‘; Q0
o N oo—N\ N~\< ‘6 NW"~90
N/ /
Method6 \ N Method7 /
————> —> I
or Method 63 /
I or Method 73 N
‘N II
N Compound I—1
Method 1:
To a solution of tetrahydropyran-4—amine (100 g, 988.7 mmol) in MeOH (3.922 L)
was added 4—(diethoxymethy1)benzaldehyde (196.1 g, 941.6 mmol) over 2 min at RT. The
reaction mixture was stirred at RT for 80 min, until the aldimine formation was complete (as
seen by NMR). NaBH4 (44.49 g, 1.176 mol) was carrefully added over 45 min, maintaining
the temperature n 24 °C and 27 °C by mean of an ice bath. After 75 min at RT, the
reaction has gone to tion. The reaction mixture was quenched with 1M NaOH (1 L).
The reaction mixture was partitioned between brine (2.5 L) and TBDME (4 L then 2 x 1 L).
The organic phase was washed with brine (500 mL) and concentrated in vacuo. The crude
mixture was lved in DCM (2 L). The aqueous phase was ted, the organic phase
was dried over MgSO4, filtered and concentrated in vacuo to give the title compound as a
yellow oil (252.99 g, 91%).
W0 2013I049726
Method 2:
A solution ofN-[[4-(diethoxymethyl)phenyl]methyl] tetrahydropyran~4-amine
(252.99 g, 862.3 mmol) and Boo anhydride (191.9 g, 202.0 mL, 879.5 mmol) in DCM (2.530
L) was cooled down to 3.3 °C. Et3N (89.00 g, 122.6 mL, 879.5 mmol) was added over 4 min,
keeping the al temperature below 5 °C. The bath was d 45 min after the end of
the addition. And the on mixture was stirred at RT ght. The reaction mixture was
sequentially washed with 0.5 M citric acid (1 L), saturated NaHCO3 solution (1 L) and brine
(1 L). The c phase was dried (MgSO4), d and concentrated in vacuo to give a
colourless oil (372.38 g, 110%). 1H NMR (400.0 MHz, DMSO); MS (ES+)
Method 3:
tert-butylN—[[4-(diethoxymethyl)phenyl]methyl]-N-tetrahydropyran—4—y1—carbamate
(372.38 g, 946.3 mmol) was dissolved in THF (5 L) and water (500 mL). Hydroxylamine
hydrochloride (72.34 g, 1.041 mol) was added in one portion and the reaction mixture was
stirred overnight at RT. The reaction mixture was partitioned between DCM (5 L) and water.
The combined organic t was washed with water (1L x 2). The organic phase was
concentrated in vacuo to a volume of about 2L. The organic layer was dried over MgSO4,
filtered and concentrated in vacuo to give a sticky colourless oil that crystallized on standing
under vacuo. (334.42g, 106%). 1H NMR (400.0 MHz, CDC13); MS (ES+)
Method 4:
tert—butylN—[[4—[(E)—hydroxyiminomethyl]phenyl]methyl]-N-tetrahydropyran-4—yl—
carbamate (334.13 g, 999.2 mmol) was dissolved in isopropyl acetate (3.0 L) (the mixture
was warmed to 40 °C to allow all the solids to go into solution). N—chlorosuccinimide (140.1
g, 1.049 mol) was added portionwise over 5 min and the reaction mixture was heated to 55
°C (external block temperature). After 45 min at 55°C The reaction had gone to completion.
The reaction mixture was cooled down to RT. The solids were d off and rinsed with
Isopropyl acetate (1 L). ed organic extract was sequentially washed with water (1.5
L, 5 times) and brine, dried over MgSO4, filtered and concentrated in vacuo to give a viscous
yellow oil (355.9 g; 96%). 1H NMR (400.0 MHz, CDC13); MS (ES+)
Method 5:
EN (76.97 g. 106.0 mL, 760.6 mmol) was added over 20 minutes to a solution of
tert—butyl N—(5-bromo—3 -ethynyl-pyrazin—Z—yl)—N-tert-butoxycarb0nyl-carbamate (233.0 g,
585.1 mmol) and teit-butyl N-[[4-[(Z)-C-chloro-N-hydroxy-carbonimidoyl]phenyl]methyl]-
N—tetrahydropyranyl-carbamate (269.8 g, 731.4 mmol) in DCM (2.330 L) at RT. During
W0 2013f049726
addition of triethylamine, the exotherm was stabilised by cooling the mixture in an ice bath,
then the reaction mixture was gradually warmed up to RT and the mixture was stirred at RT
overnight. The reaction mixture was sequentially washed with water (1.5 L, 3 times) and
brine. The organic extract was dried over MgSO4, filtered and partially concentrated in
vacuo. e (1.5L) was added and the concentration was continued yielding 547.63 g of a
yellow—orange solid.
542.12 g was taken up into ~2 vol (1 L) of ethyl acetate. The mixture was heated to
74—75 °C ally and d until all the solid went into solution. Heptane (3.2 L) was
added slowly via addition funnel to the hot solution keeping the internal temperature between
71 °C and 72 °C . At the end of the addition, the dark brown solution was seeded with some
tallised product, and the reaction mixture was allowed to cool down to RT without any
stirring to crystallise O/N. The solid was filtered off and rinsed with heptane (2 x 250 mL),
then dried in vacuo to yield 307.38 g of the title product (72 %). %). 1H NMR (400.0 MHz,
CDCl3); MS (ES+)
Method 6:
tert~butyl N—[[4-[5-[3-[bis(tert—butoxycarbonyl)amino]bromo-pyrazinyl]
isoxazol—3-yl]phenyl]methyl]-N-tetrahydropyran-4—yl-carbamate (303 g, 414.7 mmol) and 2-
methyl[4-(4,4,5,5-tetramethyl-l ,3,2-dioxaborolanyl)pyridyl] propanenitrile (112.9 g,
414.7 mmol) were ded in MeCN (2 L) and H20 (1 L). Na2C03 (414.7 mL of 2 M,
8294 mmol) followed by Pd[P(tBu)3]2 (21.19 g, 41.47 mmol) were added and the reaction
mixture was degassed with N2 for 1 h. The reaction mixture was placed under a nitrogen
atmosphere and heated at 70 °C (block temperature) for 4 h nal temperature fluctuated
between 60 °C and 61 °C). The reaction was cooled down to room ature and stirred at
RT ght. The reaction mixture was partitioned between EtOAc (2 L) and water (500
mL). The ed organic extract was washed with brine (500 mL), filtered through a short
pad of celite and concentrated under reduced pressure to a volume of about 3 L. The solution
was dried over MgSO4, filtered and partially concentrated in vacuo. iPrOH (1.5 L) was added
and the solvent was removed in vacuo to yield the desired product as a light brown foam (405
400 g was taken up into ~5 vol (2 L) of iPrOH and the mixture was heated to 80 °C
until all the solid went into solution. The dark brown solution was seeded, and the reaction
mixture was allowed to slowly cool down to RT overnight. The solid was filtered off and
rinsed with iPrOH (2 x 250 mL) and Petroleum ether (2x200 mL). The resulting solid was
W0 2013/‘049726 PCT/U82012/058127
slurried in petroleum ether (2.5 L), filtered off and dried in vacuo. The resulting solid was
dissolved in DCM (2.5 L) and stirred slowly for 1 h with 30 g of SPM32 ( 3-mercaptopropyl
ethyl sulfide silica). The silica was filtered through a pad of florisil and rinsed with DCM.
The procedure was repeated twice, then the DCM solution was concentrated in vacuo to give
23 8.02 g of a light yellow solid.
Method 7:
tert—butyl N—[[4-[5-[3-[bis(tert—butoxycarbonyl)amino]~6—[2-(l—cyanomethyl—
—4—pyridyl]pyraziny1]isoxazol—3-yl]phenyl]methyl]~N—tetrahydropyran-4—yl—
carbamate (238 g, 299.0 mmol) was dissolved in DCM (2.380 L). TFA (500 mL, 6.490 mol)
was added at RT over 3 min, The reaction mixture was stirred at RT for 3.5 h. The reaction
e was concentrated under reduced re then azeotroped with heptane (2x300ml).
The oil was then slurried in abs. EtOH (2.5 L) and filtered a
. The solid was dissolved in
mixture of ethanol (1.190 L) and water (1.190 L). potassium carbonate (124.0 g, 897.0 mmol)
in water (357.0 mL) was added to the solution and the mixture was stirred at RT overnight.
The solid was filtered off, was washed with water (2.5 L), and dried at 50 °C in
vacuo to give 108.82 g of the title compound (Compound 1—1) as a yellow powder. (73 %)
Methods 6a and 7a
Os ,0
B Method 6a
BocxN,Bo%'N\NW /
\N ON
I ‘Boc
._.._..__.______..
0 1. cat. Pd(dtbpf)Clz, PhCHa,
aq. K2003
2, crystallization
N ,
\ 1.TFA
\ N \
N~Boc DCM I NH
/ N /“ 6 25°C
-—-—————> (3
o 2. NaOH / O
I 90% I
\ CN
Compound 1-]
] A mixture of tert—butyl N-[[4—[5-[3-[bis(tert—butoxycarbonyl)amino]—6—bromo—
pyrazin-Z-yl] isoxazolyl]pheny1]methyl]-N-tetrahydropyranyl-carbamate (110.0 g, 151
mmol), K2CO3 (41.6 g, 301 mmol), and 2-methy1—2-[4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan—2—y1)—2-pyridy1] propanenitrile (41.0 g, 151 mmol) in toluene (770 mL) and
water (220 mL) is stirred and degassed with N2 for 30 min. at 20 °C. The st
Pd(dtbpf)Clz (1.96 g, 3.01 mmol) is added and the mixture is degassed for an additional 10
min. The mixture is heated at 70 CC until the reaction is complete. The mixture is cooled to
ambient temperature, diluted with water (220 mL), and filtered through a bed of Celite. The
organic phase is concentrated to remove most of the solvent. The concentrate is diluted With
i-PrOH (550 mL). The resultant suspension is d for at least 1 h and then the solid is
collected by ion to afford a tan powder. The solid is dissolved in e (990 mL) and
stirred with Biotage MP-TMT resin (18.6 g) for 2 h at ambient temperature. The resin is
removed by filtration. The filtrate is concentrated then diluted with i-PrOH (550 mL) and
then re—concentratd. Add i—PIOH (550 mL) and stir for 1 h at ambient ature. Cool the
suspension to 5 °C and collect the solid by ion then dry to afford tert—butyl N—[[4—[5-[3-
[bis(tert-butoxycarbony1)amino]—6—[2—(1-cyano—1—methyl—ethyl)—4—pyridyl]pyrazin~2-
yl]isoxazoly1]pheny1]methy1]-N-tetra'hydropyran—4-yl-carbamate (Compound 1-1) (81.9 g;
68%, yield, 98.7 area % purity by HPLC) as a cream-colored powder.
Form Chan 6 to Corn ound I-l-HCl-1.5 H20
,N -N
\ \
N \ N \
| N”
/N 1MHC| l N”
0 /N
———-————>
/ o / (j;
-1.5H20 o
\ CN \N CN
] A suspension of tert—butyl N-[[4—[5-[3—[bis(tert-butoxycarbony1)amino]-6—[2-(1-
cyanomethyl—ethyl)—4—pyridyl]pyrazin-2~yl]isoxazol—3 -yl]pheny1]methy1]—N-
tetrahydropyrany1—carbamate (Compound 1-1) (36.0 g, 72.6 mmol) in CH3CN (720 mL) is
stirred at ambient temperature (20 °C) in a flask equipped with mechanical stirring. A 1 M
aqueous solution of HCl (72.6 mL; 72.6 mmol) is added. The suspension is stirred at ambient
temperature for 20 h. The solid is collected by filtration. The filter-cake is washed with
CH3CN (3 x 50 mL) then dried under vacuum with high humidity for 2 h to afford
Compound 1—1°HC1°1.5 H20 (30.6 g; 74%) yield, 98.8 area % purity by HPLC) as a yellow
powder. .1H NMR (400 MHz, DMSO) 5 9.63 (d, J= 4.7 Hz, 2H), 9.05 (s, 1H), 8.69 (d, J =
.2 Hz, 1H), 8.21 (s, 1H), 8.16 — 8.03 (m, 3H), 7.84 (t, J= 4.1 Hz, 3H), 7.34 (br s, 2H), 4.40 —
4.18 (m, 2H), 3.94 (dd, J: 11.2, 3.9 Hz, 2H), 3.32 (t, J: 11.2 Hz, 3H), 2.17 — 2.00 (m, 2H),
1.81 (s, 6H), 1.75 (dd, J= 12.1, 4.3 Hz, 2H).
Exam 1e 2: S nthesis of 3-
- 4-iso r0 lsulfonvl hen l razin—Z—amine Com ound II-l '
(B0C)2N
NH; NHZ TMS (BOC)2N ms I
NJ§r3r ¢ //
N \ 800 N \ 1)SUZUKi
K7“! mp RNSonogashira Protection {KN 2)TMS Removal
Br Br Br
i H Hi W
\o \o \o \o
\ \ O O
0 Amide Formation 0 Reduction \0 ggidion \O Y K
—-——————> ———————>
o\ NH2 NH2 ——_——> NH
D n D D
0 0
V vii viii
0 HOW“ H0\|N
\ 0 0 ‘ O O
tion. O Y 7< Oxtme Formation. . O 0 OrOOXll'ne Y \'< cm
Formation Y \'<
N\ N\ N\
D D D D D D
u fl
O=< + ””2 0'N
\ HN—N
\ D
N] \N D l
D /N
[3+2] cycloaddilon Deproteclion
~—-——-—> -——>
0=S=O
xii II-l
Step 1: 5-Bromo-3—((trimethylsilyl)ethynyl)pyrazinamine
NHZ TMS
(Trimethylsilyl)acetylene (1.845 g, 2.655 mL, 18.78 mmol) was added dropwise
to a solution of bromopyrazin—2—amine (compound 1) (5 g, 19.77 mmol) in DMF (25
mL). Triethylamine (10.00 g, 13.77 mL, 98.85 mmol), copper(1) iodide (451.7 mg, 2.372
mmol) and Pd(PPh3)4 (1.142 g, 0.9885 mmol) were then added and the resulting solution
d at RT for 30 minutes.The reaction mixture was diluted with EtOAc and water and the
layers separated. The aqueous layer was extracted further with EtOAc and the combined
organic layers washed with water, dried (MgSO4) and concentrated in vacuo. The e was
purified by column chromatography eluting with 15% EtOAc/Petroleum ether to give the
product as a yellow solid (3.99 g, 75% Yield). 1H NMR (400.0 MHZ, DMSO) 3 0.30 (9H, s),
8.06 (II-I, 5); MS (ES+) 271.82.
Step 2: tert—Butyl N-tert-butoxycarbonyI-N-[S-br0m0((trimethylsilyl)ethyynyl)
pyrazin-Z-yl]carbamate
(BOC)2N TMS
5—Br0mo—3-(2-t1imethylsilylethynyl)pyrazin-2—amine (2.85 g, 10.55 mmol) was
dissolved in DCM (89.06 mL) and treated with Boc anhydride (6.908 g, 7.272 mL, 31.65
mmol) followed by DMAP (128.9 mg, 1.055 mmol). The reaction was allowed to stir at
ambient temperature for 2 hours. The mixture was then diluted with DCM and NaHC03 and
the layers separated. The aqueous layer was extracted further with DCM, dried (MgSQ4),
d and concentrated in vacuo. The resultant residue was purified by column
tography eluting with dichloromethane to give the d product as a colourless oil
(4.95g, 99% Yield). 1H NMR (400.0 MHZ, DMSO) 5 0.27 (9H, s). 1.42 (18H, s), 8.50 (1H,
); MS (ES+) 472.09.
Step 3: tert-Butyl N—(3-ethynyl-5—(4—(isopropylsulfonyl)phenyl)pyrazin—2—yl)N-
toxycarbonyl—carbamate tert—butyl
(BOC)2N
O=S=O
N— [5-Bromo(2-trimethylsi1ylethynyl)pyrazinyl]-N-
tertbutoxycarbonylcarbamate (3 g, 6.3 77 mmol) and (4-isopropylsulfonylphenyl)boronic acid
(1.491 g, 6.536 mmol) were dissolved in MeCN/water (60/12 mL). K3PO4 (2.706 g, 12.75
mmol) was added and the reaction mixture was ed with a flow of nitrogen (5 cycles).
Bu)3]2 (162.9 mg, 0.3188 mmol) was added and the resulting mixture was stirred at
room temperature for 1h. The reaction mixture was poured quickly into a mixture of ethyl
e (500 mL), water (90 mL) and 1% aqueous sodium metabisulphite at 4 °C, shaken well
and the layer separated. The organic fraction was dried over MgSO4, filtered and the filtrate
was treated with 3-mercaptopropyl ethyl sulphide on silica (0.8mmol/g, 1 g), pie-absorbed
onto silica gel then purified by column chromatography on silica gel g with 30—40%
EtOAc/petroleum ether. The ts were concentrated in vacuo to leave the t as a
yellow viscous oil that was triturated with petroleum ether to yield the product as beige
crystals (1.95 g, 61% Yield); 1H NMR (400 MHz, DMSO) 5 1.20 (m, 6H), 1.39 (s, 18H),
3.50 (m, 1H), 5.01 (s, 1H), 8.03 (m, 2H), 8.46 (m, 2H) and 9.37 (s, IH).
Step 4: 4-(Dimethoxymethy1)benzamide
A mixture of methyl 4—(dimethoxymethyl)benzoate (3.8 g, 18.08 mmol) and 7M
NH3 in MeOH (30 mL of 7 M, 210.0 mmol) in a sealed tube was heated at 110 °C for 22
hours. A further portion of 7M NH3 in MeOH (20 mL of 7 M, 140.0 mmol) was added and
the reaction heated at 135 °C for 23 hours. The reaction was cooled to ambient temperature
and the solvent removed in vacuo. The residue was re—submitted to the reaction conditions
(7M NH3 in MeOH (30 mL of 7 M, 210.0 mmol) at 115 °C) for a further 16 hours. The
solvent was removed in vacuo and the e tritruated from Et20. The resultant precipitate
was ed by filtration to give the sub—title compound as a white solid (590 mg, 17% yield).
The filtrate was purified by column chromatography (ISCO Companion, 40 g column, eluting
with 0 to 100% EtOAc/Petroleum Ether to 10% MeOH/EtOAc, loaded in EtOAc/MeOH) to
give a further protion of the tle product as a white solid (225 mg, 6% Yield). Total
PCT/U52012/058127
isolated (815 mg, 23% Yield); 1H NMR (400 MHz, DMSO) 5 3.26 (s, 6H), 5.44 (s, 1H), 7.37
(s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.84 - 7.91 (m, 2H) and 7.98 (s, 1H) ppm; MS (ES+) 196.0.
Steo 5: erio—[4-(dimethoxymethyl)phenyl]methanamine
LiDH4 (12.52 mL of 1 M, 12.52 mmol) was added dropwise to a stirred solution of 4-
(dimethoxymethyl)benzamide (815 mg, 4.175 mmol) in THF (20 mL) at 0 °C under an
atmosphere of nitrogen. The reaction was heated at reflux for 16 hours then cooled to ambient
temperature. The reaction was quenched by the sequential addition of D20 (1 mL), 15%
NaOH in D20 (1 mL) and D20 (4 mL). The ant solid was removed by filtration and
washed with EtOAc. The e was concentrated in vacuo and the residue dried by
azeotropic distillation with e (x 3) to give the sub-title compound as a yellow oil (819
mg) that was used without further purification; 11-1 NMR (400 MHz, DMSO) 5 3.23 (3, 61-1),
.36 (s, 1H) and 7.30 - 7.35 (m, 4H) ppm; MS (ES+) 167.0.
Step 6: tert-Butyl N-[dideuterio—[4-(dimethoxymethyl)phenyl]methyl]carbamate
\o OYOKNH
D D
viii
Eth (633.7 mg, 872.9 pL, 6.262 mmol) was added to a stirred suspension of
dideuterio—[4-(dimethoxymethyl)pheny1]methanamine (765 mg, 4.175 mmol) in THF (15
mL) at 0 °C. The reaction was allowed to stir at this ature for 30 minutes then Boczo
(956.8 mg, 1.007 mL, 4.384 mmol) was added in portions. The reaction was d to warm
to ambient temperature and stirred for 18 hours. The solvent was removed in vacuo and the
residue was purified by column chromatography (ISCO Companion, 120 g column, eluting
with 0 to 50% EtOAc/Petroleum Ether, loaded in DCM) to give the sub-title product as a
colourless oil (1.04 g, 88% Yield); 1H NMR (400 MHz, DMSO) 6 1.40 (s, 9H), 3.23 (s, 6H),
.36 (s, 1H), 7.24 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H) and 7.38 (s, 1H) ppm.
Step 7: tert-Butyl N-[dideuterio—[4-(dimethoxymethyl)phenyl]methyl]-N-methyl—
carbamate
\o 0Y07<N
D D ‘
LiHMDS (1M in THF) (1.377 mL of l M, 1.377 mmol) was added dropwise to
a sittred solution of tert—butyl N—[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]carbamate
(300 mg, 1.059 mmol) in THF (5 mL) at —78 °C. The solution was stirred at this temperature
for 10 minutes then thane (225.4 mg, 98.86 uL, 1.588 mmol) was added se and
the mixture allowed to warm to ambient ature over 1 hour. The reaction was again
cooled to ~78 °C and LiHMDS (1M in THF) (635.4 uL of 1 M, 0.6354 mmol) was added.
After 10 minutes iodomethane (105.2 mg, 46.14 uL, 0.7413 mmol) was added and the
reaction allowed to warm to ambient temperature over 6 hours. The mixture was diluted with
EtOAc and the organic layer washed with saturated aqueous NaHC03 (x 2), brine (x 1), dried
(MgSO4) filtered and concentrated in vacuo. The residue was purified by column
chromatography (ISCO Companion, 24 g column, eluting with 0 to 30% EtOAc/Petroleum
Ether, loaded in DCM) to give the sub-title product as a colourless oil (200 mg, 63% Yield);
1H NMR (400 MHz, DMSO) 6 1.41 (d, J = 27.7 Hz, 9H), 2.76 (s, 3H), 3.24 (s, 6H), 5.37 (s,
1H), 7.23 (d, J = 7.9 Hz, 2H) and 7.37 (d, J = 8.0 Hz, 2H) ppm.
Step 8: tert—Butyl euterio-[4-[hydroxyiminomethyl]phenyl]methyl]-N—
(methyl)carbamate
OYOKN
D D
Hydroxylamine hydrochloride (51.15 mg, 0.7361 mmol) was added to a d
solution of tert-butyl N-[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]-N-methyl-
carbamate (199 mg, 0.6692 mmol) in THF (10 mL)/water (1.000 mL) and the on
d to stir at ambient temperature for 4 hours. The reaction was partitioned between
DCM and brine and the layers separated. The aqueous layer was extracted with DCM (x 2)
and the combined organic extracts washed with brine (x 1), dried (MgSO4), filtered and
WO 49726 PCT/U52012/058127
concentrated in vacuo to give the sub—title compound as a white solid (180 mg, 100 % Yield);
1H NMR (400 MHz, DMSO) 8 1.41 (d, J = 24.6 Hz, 9H) 2.76 (s, 3H), 7.25 (d, J = 8.1 Hz,
2H), 7.58 (d, J = 8.0 Hz, 2H), 8.13 (s, 1H) and 11.20 (s, 1H) ppm; MS (ES+) 211.0 (M-Boc).
Step 9: tert-Butyl N-[[4-[chloro—N—hydroxy-carbonimidoyl]phenyl]-dideuterio-methyl]-
N—methyl—carbamate
D D
tert—Butyl N~[dideuterio-[4-[hydroxyiminomethyl]phenyl]methyl]—N—
(methyl)carbamate (178 mg, 0.6683 mmol) in DMF (2 mL) was d with NCS (89.24 mg,
0.6683 mmol) and the reaction warmed to 65 °C for 1 hour. The reaction was cooled to
ambient temperature and diluted with water. The mixture was extracted with EtOAc (x 2) and
the combined organic extracts washed with brine (x 4), dried (MgSO4), filtered and
concentrated in vacuo to give the sub-title compound as a white solid (188 mg, 94% Yield);
1H NMR (400 MHZ, DMSO) 8 1.42 (d, J = 24.7 Hz, 9H), 2.78 (s, 3H), 7.32 (d, J = 8.4 Hz,
2H), 7.78 (d, J = 8.2 Hz, 2H) and 12.36 (s, 1H) ppm.
Step 10: tert-Butyl N-[[4-[5—[3-[bis(tert-butoxycarbonyl)amin01—6-(4-
isopropylsulfonylphenyl)pyrazin-2—yl]isoxazol—S-yl]phenyl]-dideuteri0-methyl]-N-
methyl-carbamate
Oz< ‘6
(BOC)2NWW041 N\
Eth (36.31 mg, 50.01 uL, 0.3588 mmol) was added dropwise to a stirred
on of tert-butyl N-teit-butoxycarbonyl-N-[3 -ethyny1-5~(4-
isopropylsulfonylphenyl)pyrazin—2—yl]carbamate (150 mg, 0.2990 mmol) and tert—Butyl N-
[[4-[chloro—N-hydroxy-carbonimidoyl]phenyl]—dideuterio—methyl]~N~methyl-carbamate
(89.93 mg, 0.2990 mmol) in ous THF (3 mL) and the reaction mixture heated at 65 °C
for 3 hours. The reaction mixture was cooled to ambient temperature and diluted with
EtOAc/brine. Water was added until the aqueous layer became clear and the layers were
ted. The s layer was extracted with EtOAc (x 1) and the combined organic
extracts were washed with brine (x 1), dried (MgSO4), filtered and concentrated in vacuo.
The residue was purified by column chromatography (ISCO ion, 40 g column,
elueting with 0 to 30% EtOAc/Petroleum Ether, loaded in DCM) to give the sub-title product
as a White solid (134 mg, 59% Yield); 1H NMR (400 MHZ, DMSO) 6 1.22 (d, J = 6.8 Hz,
6H) 1.32 (s, 18H), 1.43 (d, J = 23.1 Hz, 9H), 2.82 (s, 3H), 3.56 (pent, 1H), 7.43 (d, J = 8.3
Hz, 3H), 8.02 - 8.03 (m 3H), 8.06 — 8.11 (m, 2H), 8.62 — 8.67 (m, 2H) and 9.51 (s, 1H) ppm;
MS (ES+) 666.2 (M-Boc).
Step 11: 3-[3-[4-[Dideuterio(methylamino)methyl]phenyl]isoxazol—S—ylj-S-(4-
isopropylsulfonylphenyl)pyrazin—2-amine (compound II-l)
II—l
3M HCl in MeOH (1.167 mL of 3 M, 3.500 mmol) was added to a stirred
solution of tert-butyl N~[[4-[5—[3—[bis(tert—but0xycarbonyl)amino]—6-(4-
isopropylsulfonylphenyl)pyrazin—2—y1]isoxazol~3-yl]phenyl]-dideuterio-methyl]—N—methyl—
carbamate (134 mg, 0.1750 mmol) in DCM (5 mL) and the reaction heated at reflux for 16
hours. The reaction was cooled to ambient temperature and the resultant precipitate was
ed by filtration and dried under vacuum at 40 °C to give the di-HCl salt of the title
compound as a yellow solid (58.8 mg, 62% Yield); 1H NMR (400 MHz, DMSO) 5 1.20 (d, J
= 6.8 Hz, 6H), 2.60 (t, J = 5.4 Hz, 3H), 3.48 (hept, J = 6.8 Hz, 1H), 7.22 (br s, 2H), 7.69 —
7.75 (m, 2H), 7.85 (s, 1H), 7.92 - 7.99 (m, 2H), 8.08 — 8.15 (m, 2H) 8.37 - 8.42 (m, 2H),
8.97 (s, 1H) and 9.10 (d, J = 5.8 Hz, 2H) ppm; MS (ES+) 466.2.
D ‘N
0 0 D D
\ \i/ I O\i/D
\O Y K .
Alkylation O Oxime Formation Chloroqxume
—_> ion
NH N O ———————>
N 0
D o D D 701/ \i< a u 1)], W<
viii xiii xiv
A D
0 NH; 0"
\ HN 6 D
O=<N D \ D
(BOC)2N o—N N \ 0
HOW \\ 3D I
I D
D NI \ D
CI \1/D [3+2] dditon /N ection
-———————-—’ -
”Y°j<
O=S=O
xv xvi “-2
Step 1: tert—Butyl N-[dideuterio- [4-(dimethoxymethyl)phenyl] methyl]
(trideuteriomethyl)carbamate
LiHMDS (1M in THF) (1.181 mL of 1 M, 1.181 mmol) was added dropwise to
a sittred solution of tert—butyl N—[dideuterio-[4—(dimethoxymethy1)phenyl]methyl]carbamate
(300 mg, 1.059 mmol) in THF (5 mL) at ~78 °C. The solution was stirred at this temperature
for 30 minutes then trideuterio(iodo)methane (198.0 mg, 84.98 uL, 1.366 mmol) was added
dropwise and the mixture allowed to warm to ambient temperature over 21 hours. The
reaction was again cooled to -78 °C and a r portion of LiHMDS (1M in THF) (635.4
pL of 1 M, 0.6354 mmol) was added. After 15 minutes more trideuterio(iodo)methane (76.75
mg, 32.94 pL, 0.5295 mmol) was added and the reaction allowed to warm to ambient
ature over 5 hours. The mixture was diluted with EtOAc and the organic layer washed
with saturated aqueous NaHC03 (x 2), brine (x 1), dried (MgSO4) filtered and concentrated in
vacuo. The residue was purified by column chromatography (ISCO Companion, 24 g
column, eluting with 0 to 30% EtOAc/Petroleum Ether, loaded in DCM) to give the sub-title
product as a colourless oil (213 mg, 67% Yield); 1H NMR (400 MHz, DMSO) 5 1.36 - 1.42
(m, 9H) 3.22 (s, 6H), 5.35 (s, 1H), 7.21 (d, J = 7.8 Hz, 2H) and 7.35 (d, J = 7.7 Hz, 2H) ppm.
PCT/U32012/058127
Step 2: tert-Butyl N—[dideuterio—[4-[hydroxyiminomethyl]phenyl]methyl]-N—
(trideuteriomethyl)carbamate
D. D
o 7<
Hydroxylamine hydrochloride (53.95 mg, 0.7763 mmol) was added to a stirred
solution of tert—butyl N—[dideuterio-[4-(dimethoxymethyl)phenyl]methyl]—N-
uteriomethyl)carbamate (212 mg, 0.7057 mmol) in THF (10 mL)/water (1.000 mL) and
the reaction allowed to stir at t temperature for 22 hours. The reaction was partitioned
between DCM and brine and the layers separated. The aqueous layer was extracted with
DCM (x 2) and the combined organic extracts washed with brine (x 1), dried (MgSO4),
filtered and trated in vacuo to give the sub—title compound as a white solid (190 mg,
100% Yield). ); 1H NMR (400 MHz, DMSO) 6 1.41 (d, J = 24.2 Hz, 9H) ), 7.25 (d, J = 8.1
Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 8.13 (s, 1H) and 11.20 (s, 1H) ppm.
Step 3: tert-Butyl N-[[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]-dideuterio-methyl]-
N-(trideuteriomethyl)carbamate
HO\N
I D D
a +
N\n’OK
D D
tert—Butyl N—[dideuterio-[4-[hydroxyiminomethyl]phenyl]methyl]—N—
uteriomethy1)carbamate (190.0 mg, 0.7054 mmol) in DMF (2 mL) was treated with
NCS (94.19 mg, 0.7054 mmol) and the reaction warmed to 65 °C for 1 hour. The reaction
was cooled to ambient temperature and diluted with water. The mixture was extracted with
EtOAc (x 2) and the combined organic extracts washed with brine (x 4), dried (MgSO4),
filtered and trated in vacuo to give the sub-title compound as a white solid (198 mg,
93% Yield); 1H NMR (400 MHZ, DMSO) 5 1.41 (d, J = 26.0 Hz, 9H), 7.32 (d, J = 8.3 Hz,
2H), 7.78 (d, J = 8.2 Hz, 2H) and 12.36 (s, 1H) ppm.
W0 2013f049726 PCT/U52012/058127
Step 4: tert-Butyl N—[ [4-[5-[3-[bis(tert-butoxycarbony1)amino](4-
isopropylsulfonylphenyl)pyrazin—2-y1]isoxazol—S—yl]phenyl]-dideuterio—methyl]-N-
(trideuteriomethyl)carbamate
0% D
(Boc)2N O'N\ N40
\ D
N \ D
I D
o=s=o
EN (36.31 mg, 50.01 uL, 0.3588 mmol) was added dropwise to a stirred
solution of tert-butyl N—tert—butoxycarbonyl-N-[3 —ethyny1-5—(4—
isopropylsulfonylphenyl)pyrazin-2~y1]carbamate (150 mg, 0.2990 mmol) and tert—butyl N-
hloro-N—hydroxy—carbonimidoyl]phenyl]-dideuterio—methyl]—N-
(trideuteriomethyl)carbamate (90.84 mg, 0.2990 mmol) in anhydrous THF (3 mL) and the
reaction mixture heated at 65 °C for 3.5 hours. The reaction mixture was cooled to ambient
temperature and diluted with EtOAc/brine. Water was added until the aqueous layer became
clear and the layers were separated. The aqueous layer was ted with EtOAc (x l) and
the combined organic ts were washed with brine (x 1), dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by column chromatography (ISCO
ion, 40 g column, elueting with 0 to 35% EtOAc/Petroleum Ether, loaded in DCM)
to give the sub—title product as a white solid (158 mg, 69% Yield); 1H NMR (400 MHz,
DMSO) 8 1.22 (d, I = 6.8 Hz, 6H) ), 1.44 (d, J = 22.0 Hz, 9H), 3.56 (dt, J = 13.5, 6.7 Hz, 2H),
7.43 (d, J = 8.2 Hz, 3H), 8.02 (d, J = 6.9 Hz, 2H), 8.08 (d, J = 8.7 Hz, 2H), 8.65 (d, J = 8.8
Hz, 2H) and 9.51 (s, 1H) ppm; MS (ES+) 669.3 (M—Boc).
Step 5: 3—[3-[4-[dideuteri0-(trideuteriomethylamino)methyl]phenyl]isoxazol—S—yl]~5-(4-
isopropylsulfonylphenyl)pyrazin—2-amine (compound 11-2)
NH2 0"‘{ HN‘é o
\ D
N \ o
i D
o=s=o
11-2
] 3M HCl in MeOH (1.361 mL of 3 M, 4.084 mmol) was added to a stirred
solution of tert—butyl N-[[4—[5-[3—[bis(tert-butoxycarbonyl)amino]—6—(4-
isopropylsulfonylphenyl)pyrazin—2—yl]isoxazol-3 ~yl]phenyl]-dideuterio-methyl]~N—
(trideuteriomethyl)carbamate (157 mg, 0.2042 mmol) in DCM (5 mL) and the reaction heated
at reflux for 16 hours. The reaction was cooled to ambient temperature and the resultant
precipitate was isolated by filtration and dried under vacuum at 40 °C to give the di-HCl salt
of the title compound as a yellow solid (72.5 mg, 66% Yield); 1H NMR (400 MHz, DMSO)
6 1.20 (d, J = 6.8 Hz, 6H), 3.48 (dq, J = 13.6, 6.7 Hz, 1H), 7.21 (s, 2H), 7.68 — 7.78 (m, 2H),
7.85 (s, 1H), 7.91 - 8.42 (m, 2H), 8.96 (s, 1H) and
- 7.99 (m, 2H), 8.08 - 8.13 (m, 2H), 8.36
9.14 (s, 2H) ppm; MS (ES+) 469.1.
Exam 1e 4: S nthesis of 5- 4—iso r0 lsulfon l hen 1 3- 4-
trideuteriometh lamino meth l hen lisoxazol-S- l razin-Z-amine
iCompound 11-3)
0 o
\OJKQ/NW 800 \o Reduction
Protection midan/ Alkylaiion
0% D+\:/OOK
xvii xviii xix
m/\©\l:\1:;roD\‘/D HO‘N
| D
l D\T,D
Oxidation NYOK Oxime Formation N 0 $333:ng
o l<
xx xxi xxii
3< D
o ””2 0’N\ HN+D
ECU»? D D
N 0’” 0%
\ n+0 NW,
\ ’N
“‘ D
I .
ycloadditon / N Deprotec’tion
————————>
xxiii xxiv A II-3
O=S=O
W0 2013f049726 PCT/U82012/058127
Step 1: Methyl rt—butoxycarbonylamino)methyl]benzoate
xviii
Et3N (1.882 g, 2.592 mL, 18.60 mmol) was added to a stirred suspension of
methyl 4—(aminomethyl)benzoate (Hydrochloric Acid (1)) (1.5 g, 7.439 mmol) in THF (20
mL) at 0 °C. The reaction was allowed to stir at this ature for 30 minutes then B0020
(1.705 g, 1.795 mL, 7.811 mmol) was added in ns. The reaction was allowed to warm
to ambient temperature and stirred for 18 hours. The mixture was diluted with EtOAc. The
organic layer was washed with 1M aqueous HCl (x 2), saturated aqueous NaHC03 (x 2) and
brine (x 1). The organic layer was dried (MgSO4), filtered and concentrated in vacuo to give
the sub-title compound as a white solid that was used without further purification (1.93 g,
98% Yield); 1H NMR (400 MHz, DMSO) 5 1.40 (s, 9H), 3.85 (s, 3H), 4.20 (d, J = 6.1 Hz,
2H), 7.38 (d, J = 8.2 Hz, 2H), 7.49 (t, l = 6.1 Hz, 1H) and 7.92 (d, J = 8.2 Hz, 2H) ppm; MS
(ES+) 251.1 (M-Mc).
Step 2: Methyl 4-[[tert-butoxycarbonyl(trideuteriomethyl)amino]methyl]benzoate
LiHMDS (1M in THF) (8.112 mL of 1 M, 8.112 mmol) was added dropwise to
a stirred solution of methyl 4-[(tert—butoxycarbonylamino)methyl]benzoate (1.93 g, 7.275
mmol) in THF (10 mL) at —78 °C. The solution was stirred at this temperature for 30 minutes
then trideuterio(iodo)methane (1.360 g, 9.385 mmol) was added dropwise and the mixture
allowed to warm to t temperature over 3 hours. The reaction was cooled to —78 °C and
a r n of LiHMDS (1M in THF) (2.182 mL of 1 M, 2.182 mmol) was added. After
minutes a further n of trideuterio(iodo)methane (527.4 mg, 3.638 mmol) was added
and the reaction allowed to warm to ambient temperature over 17 hours. The mixture was
diluted with EtOAc and the organic layer washed with saturated aqueous NaHCO; (x 2),
brine (x 1), dried (MgSO4) filtered and concentrated in vacuo. The residue was purified by
PCT/USZ012/058127
column chromatography (ISCO Companion, 120 g column, eluting with 0 to 30%
EtOAc/Petroleum Ether, loaded in DCM) to give the tle product as a pale yellow oil
(1.37 g, 67% Yield); 1H NMR (400 MHz, DMSO) 5 1.38 (d, J = 44.2 Hz, 9H), 3.83 (s, 3H),
4.43 (s, 2H), 7.33 (d, J = 8.2 Hz, 2H) and 7.94 (d, J = 8.1 Hz, 2H) ppm; MS (ES+) 268.1 (M—
Step 3: tert-Butyl N—[[4-(hydroxymethyl)phenyl]methyl]-N-
(trideuteriomethy1)carbamate
LiBH4 (158.5 mg, 7.278 mmol) was added to a stirred solution of methyl 4—
[[tert-butoxycarbonyl(trideuteriomethy1)amino]methyl]benzoate (1.37 g, 4.852 mmol) in
THF (10 mL) and the reaction warmed to 85 °C for 15 hours. A further portion of LiBH4
(158.5 mg, 7.278 mmol) was added and the reaction stirred at 65 °C for a further 7 hours. The
reaction e was cooled to ambient ature then poured onto crushed ice and whilst
stirring, ]M HCl was added se until no effervescence was observed. The mixture was
stirred for 10 minutes then saturated aqueous NaHCO3 was added until the mixture was at pH
8. The aqueous layer was extracted with EtOAc (x 3) and the combined organic extracts dried
(MgSO4), filtered and concentrated in vacuo. The residue was purified by column
chromatography (lSCO Companion, 120 g column, elueting with 0 to 100%
EtOAc/Petroleum Ether, loaded in DCM) to give the sub—title product as a colourless oil
(1.03 g, 84% Yield); 1H NMR (400 MHz, DMSO) 5 1.42 (d, J = 14.6 Hz, 9H), 4.35 (s, 2H),
4.48 (d, J = 5.7 Hz, 2H), 5.15 (t, J = 5.7 Hz, 1H), 7.18 (d, J = 7.9 Hz, 2H) and 7.30 (d, J = 7.7
Hz, 2H) ppm; MS (ES+) 181.1 (M—O’Bu).
Step 4: utyl N-[(4—formylphenyl)methyl]-N-(trideuteriomethyl)carbamate
KE:|\/| D
D\|/D
N\n/OK
] MnOz (5.281 g, 1.051 mL, 60.75 mmol) was added to a stirred solution of tert-
butyl N-[[4—(hydroxymethyl)phenyl]methyl]~N-(trideuteriomethyl)carbamate (1.03 g, 4.050
mmol) in DCM (10 mL) and the reaction stirred at ambient temperature for 20 hours. The
reaction was filtered through a pad of Celite and washed with DCM. The filtrate was
concentrated in vacuo to give the tle compound as a colourless oil (891 mg, 88%
Yield); 1H NMR (400 MHz, DMSO) 8 1.40 (d, J = 43.4 Hz, 9H), 4.48 (s, 2H), 7.43 (d, J =
8.0 Hz, 2H), 7.91 (d, J = 7.9 Hz, 2H) and 10.00 (s, 1H), ppm.
Step 5: tert—butyl N—[[4—[hydroxyiminomethyl]phenyl]methyl]-N-
(trideuteriomethyl)carbamate
xxii
Hydroxylamine (466.0 uL of 50 %w/V, 7.054 mmol) was added to a stirred
solution of tert-butyl formy1pheny1)methy1]-N—(trideuteriomethyl)carbamate (890 mg,
3.527 mmol) in ethanol (5 mL) and the reaction mixture stirred at ambient temperature for 45
minutes. The reaction mixture was trated in vacuo and the residue taken up in water
and extracted with EtOAc (x 3). The combined organic extracts were washed with brine (x
1), dried ), filtered and concentrated in vacuo. The residue was triturated from
petroleum ether and the precipitate isolated by filtration to give the sub-title product as a
white solid (837 mg, 89% ; 1H NMR (400 MHz, DMSO) 5 1.41 (d, J = 25.8 Hz, 9H),
4.38 (s, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 8.13 (s, 1H) and 11.20 (s, 1H)
ppm; MS (138+) 212.0 ).
Step 6: tert-Butyl N-[[4-[chloro-N—hydroxy—carbonimid0y1]phenyl]methyl]-N-
(trideuteriomethyl)carbamatep
xxiii
tert—butyl N—[[4—[hydroxyiminomethy1]pheny1]methyl]~N—
(trideuteriomethyl)carbamate (250 mg, 0.9351 mmol) in DMF (2.5 mL) was treated with
NCS (124.9 mg, 0.9351 mmol) and the reaction warmed to 65 °C for 1 hour. The reaction
was cooled to ambient temperature and diluted with water. The mixture was extracted with
EtOAc (x 2) and the combined organic extracts washed with brine (x 4), dried (MgSO4),
d and concentrated in vacuo to give the sub—title compound as a white solid (259 mg,
92% Yield); 1H NMR (400 MHz, DMSO) 5 1.41 (d, J = 29.6 Hz, 9H), 4.42 (s, 2H), 7.31 (d, J
= 8.3 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), and12.38 (s, 1H), ppm.
Step 7: tert-Butyl N—[[4-[5—[3-[bis(tert—but0xycarbonyl)amino](4-
isopropylsulfonylphenyl)pyrazin-Z-yllisoxazolyl]phenyl] methyl]-N-
(trideuteriomethyl)carbamate
, O=<
(800w O N\ N+D
N \ D
xxiv
[001831 Eth (48.41 mg, 66.68 uL, 0.4784 mmol) was added dropwise to a stirred
solution of tert-butyl -butoxycarbonyl-N-[3-ethynyl-5~(4-
isopropy1sulfonylphenyl)pyrazin—2—yl]carbamate (200 mg, 0.3987 mmol) and tert—butyl N—
[[4—[chloro-N-hydroxy—carbonimidoyl]phenyl]methyl]~N-(trideuteriomethyl)carbamate (120.3
mg, 0.3987 mmol) in anhydrous THF (5 mL) and the reaction mixture heated at 65 °C for 2.5
hours. The reaction e was cooled to ambient temperature and diluted with
EtOAc/brine. Water was added until the aqueous layer became clear and the layers were
separated. The aqueous layer was extracted with EtOAc (x 1) and the combined c
extracts were washed with brine (x 1), dried (MgSO4), filtered and concentrated in vacuo.
The residue was purified by column chromatography (ISCO Companion, 40 g ,
elueting with 0 to 20% EtOAc/Petroleum Ether, loaded in DCM) to give the sub—title product
as a white solid (213.5 mg, 70% Yield); 1H NMR (400 MHz, DMSO) 8 1.22 (d, J = 6.8 Hz,
6H), 1.31 (s, 18H), 1.43 (d, J = 26.2 Hz, 9H), 3.51 =
- 3.60 (m, 1H), 4.47 (s, 2H), 7.42 (d, J
8.1 Hz, 2H), 8.03 (d, J = 5.2 Hz, 3H), 8.08 (d, J = 8.6 Hz, 2H), 8.65 (d, J = 8.6 Hz, 2H) and
9.52 (s, 1H) ppm; MS (138+) 667.4 (M-Boc).
Step 8: 5-(4-Isopropylsulfonylphenyl)-3—[3—[4-[(trideuteriomethylamino)methyl]
phenyl]isoxazol-S-yl]pyrazin-Z-amine (Compound 11-3)
PCT/U32012/058127
NH2 0"‘{ HN {D
N \ D
o=s=o
11-3
3M HCl in MeOH (1.5 mL of 3 M, 4.500 mmol) was added to a stirred solution
of tert—butyl N—[[4-[5—[3—[bis(tert-butoxycarbonyl)amino](4—
isopropylsulfonylphenyl)pyrazin—2-y1]isoxazol—3—yl]phenyl]methyl]~N-
(trideuteriomethyl)carbamate (213 mg, 0.2777 mmol) in DCM (6 mL) and the reaction heated
at reflux for 15 hours. A further n of 3M HCl in M6011 (0.5 mL of 3 M, 1.500 mmol)
was added and the reaction heated at reflux for a further 7 hours. The on was cooled to
ambient temperature and the resultant precipitate was isolated by filtration and dried under
vacuum at 40 °C to give the di-HCl salt of the title compound as a yellow solid (97.6 mg,
65% Yield); 1H NMR (400 MHz, DMSO) 5 1.20 (d, J = 6.8 Hz, 6H), 3.47 (tt, J = 14.0, 6.9
Hz, 1H), 4.19 — 4.25 (m, 2H), 7.23 (s, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.85 (s, 1H), 7.95 (d, J =
8.7 Hz, 2H), 8.11 (d, J = 8.4 Hz, 2H), 8.39 (d, J = 8.7 Hz, 2H), 8.97 (s, 1H) and 9.11 (s, 2H)
ppm; MS (ES+) 467.2.
PCT/U82012/058127
tetradeuterio-l- teriometh leth n l hen l razin-Z-amine Com ound
11-41
Reductlve
/\O /\0J\©\/n S_g__>fection 4m? Oxime Formation
Amination
/O —--—---—>
xxvil
Chlorooxime (BOC)2N
. 0‘N\ N~
ion 0T0 [3+2] cycloaddlton \
—-————> N \
xxviii xxix Br xxx
Br a:
Br w
O~ ,O
Bornate
Alkylation 8 Oxidation Suzuki
SH ——>
Formation ——>
Dbz/*w<”fl 0W: D D o=s=o
D D o o
D D D
n n
xxxi xxxii xxxiii xxxiv
O NH2 o—N\ HN‘
(80an 0'N\ =<N~ N \
\ /N
Deprotection
DC):S=O
O=S=O xxxv
o o
n o 0
XXV]
2M methylamine in MeOH (288.1 mL, 576.2 mmol) was diluted with ol
(1.000 L) and stirred at ~20 °C. 4-(Dieth0xymethy1)benzaldehyde (100 g, 480.2 mmol) was
added dropwise over 1 minute and the reaction stirred at ambient temperature for 1.25 hours.
Sodium borohydride (29.07 g, 30.76 mL, 768.3 mmol) was added portionwise over 20
minutes while maintaining the temperature between 20 and 30 °C with an ice-water bath. The
reaction solution was stirred at ambient temperature ght then quenched by the dropwise
addition ofNaOH (960.4 mL of 1.0 M, 960.4 mmol) over 20 minutes. The reaction was
W0 2013/049726 2012/058127
stirred for 30 minutes and concentrated in vacuo to remove MeOH. The reaction was
partitioned with MTBE (1.200 L) and the phases separated. The organic phase was washed
with water (3000 mL), dried (N212804), and concentrated in vacuo to give the title compound
as a yellow oil (102.9 g, 96% Yield); 1H NMR (400 MHZ, CDC13) 6 1.25 (t, 6H), 2.46 (s,
3H), 3.45 ~ 3.65 (m, 4H), 3.75 (s, 2H), 5.51 (s, 1H), 7.32 (d, 2H) and 7.44 (d, 2H) ppm.
Step 2: tert-Buty] (diethoxymethyl)pheny1]methyl]-N-methyl-carbamate
LO d/
“co"N\
xxvu
A l-L glass-jacketed reactor was fitted with an overhead stirrer, thermocouple,
and chiller. A solution of l-[4-(diethoxymethyl)phenyl]-N—methyl-methanamine (80.0 g,
358.2 mmol) in DCM (480.0 mL) was stirred at 18 °C. A on of B00 anhydride (79.75 g,
83.95 mL, 365.4 mmol) in DCM (160.0 mL) was added over 10 minutes and the solution was
stirred at 20 ~ 25 °C overnight. The reaction mixture was filtered, rinsed with DCM (3 x 50
mL) and the filtrate concentrated in vacuo to afford give the title compound as a pale yellow
liquid (116.6 g, quantitative yield); 1H NMR (400 MHz, CDCl3) 5 1.25 (t, 6H), 1.49 — 1.54 (2
x s, 9H), 2.78 — 2.83 (2 x s, 3H), 3.50 4 3.66 (m, 4H), 4.42 (s, 2H), 5.49 (s, 1H), 7.22 (d, 2H)
and 7.45 (d, 2H) ppm.
Step 3: tert-Butyl [hydroxyiminomethyl]phenyl]methyl]-N-methyl-carbamate
xxviii
A biphasic solution of tert-butyl N—[[4-(diethoxymethyl)phenyl]methyl]-1V—
methyl-carbamate (50.0 g, 154.6 mmol) in 2—MeTHF (400.0 mL) and Na2S04 (100.0 mL of
%w/v, 70.40 mmol) was stirred at 8 — 10 °C in a l—L, glass—jacketed r.
Hydroxylamine hydrochloride (46.38 mL of 5.0 M, 231.9 mmol) was added and the biphasic
solution was stirred at 30 °C for 16 hours. The reaction was diluted with MTBE (200.0 mL)
and the layers separated. The organic phase was washed with water (200.0 mL), dried
(Na2S04), filtered and concentrated in vacuo. The e was diluted with heptane (200.0
PCT/U82012/058127
mL) and the resultant suspension was stirred at ambient temperature for 30 minutes. The
solid was collected by ion to give the title compound as a white solid (36.5 g, 89%
Yield); 1H NMR (400 MHz, CDC13) 5 1.50 (s, 9H), 2.88 (br s, 3H), 4.60 (s, 2H), 7.26 (d,
2H), 7.52 (d, 2H) and 8.15 (s, 1H) ppm.
Step 4: tert—Butyl N-I[4-[chloro-N-hydroxy-carbonimidoyl]phenyl]methyl]-N-methyl—
carbamate
A suspension of utyl N-[[4—[hydroxyiminomethyl]phenyl]methyl]-N—
methyl—carbamate (100 g, 378.3 mmol) in isopropyl e (1.000 L) was stirred at ambient
temperature. N-Chlorosuccinimide (53.04 g, 397.2 mmol) was added and stirred at ambient
temperature for 16 hours. The reaction was partitioned with water (500.0 mL) and the phases
separated. The organic phase was washed with water (500.0 mL) (2 x), dried (Na2804),
d and concentrated in vacuo to remove most of the solvent. Heptane (1.000 L) was
added and the mixture concentrated in vacuo to remove most of the solvent. Heptane (1.000
L) was added and the ant precipitate isolated by ion. The filter-cake was washed
with heptanc (500 mL) and air—dried to give the title compound as an off—white powder
(105.45 g, 93% Yield); 1H NMR (400 MHz, CDC13) 8 1.48 (2 x s, 9H), 2.90 (2 x s, 3H), 4.47
(s, 2H), 7.26 (d, 2H), 7.77 (d, 2H) and 8.82 (s, 1H) ppm.
Step 5: tert-Butyl N—[[4-[5-[3-[bis(tert-butoxycarbonyl)amino]bromo-pyrazin—2-
yllisoxazol—3-yl]phenyl] methyl]—N-methyl-carbamate
(BOC)2N 0’11 N\
A suspension of tert-butyl N—[[4-[chloro-N-hydroxy-
carbonimidoyl]phenyl]methyl]-N-methyl-carbamate (100.0 g, 334.7 mmol) and tert—butyl N-
tert—butoxycarbonyl-N—[3—ethynyl—5—(4—isopropylsulfonylphenyl)pyrazin—2—yl]carbamate
W0 2013f049726 PCT/USZ012/058127
(121.2 g, 304.3 mmol) in DCM (1.212 L) was stirred at ambient temperature. Triethylamine
(33.87 g, 46.65 mL, 334.7 mmol) was added in one portion and the reaction stirred at ambient
temperature for 16 hours. The reaction was partitioned with water (606.0 mL) and the phases
separated. The organic phase was washed with water (606.0 mL), dried (NaZSO4), filtered
and concentrated in vacuo to near dryness. e (3 63.6 mL) was added and the e
concentrated to about 300 mL, Further e (1.212 L) was added and the mixture heated
to 90 °C with ng. The mixture was slowly cooled to ambient temperature and stirred at
this temperature for 1 hour. The resultant precipitate was isolated by filtration and the filter—
cake washed with heptane (2 x 363.6 mL) and air-dried to give the title nd as a beige
solid (181.8 g, 90% Yield); 1H NMR (400 MHz, CDCl3) 6 1.41 (s, 18H), 1.51 (s, 9H), 2.88
(2 x s, 3H), 4.50 (s, 2H), 7.36 — 7.38 (m, 3H), 7.86 (d, 2H) and 8.65 (s, 1H) ppm.
Step 6: l-Bromo[1,2,2,2-tetradeuterio(trideutcri0methyl)ethyl]sulfanyl-benzene
xxxii
Sodium hydride (246.5 mg, 6.163 mmol) was added pofiionwise to a stirred
solution of 4—br0mobenzenethiol (compound xxxi) (970.9 mg, 5.135 mmol) in DMF (10 mL)
at 0 °C. After stirring at this temperature for 15 minutes 1,1 l ,2,3,3,3 deutetio-2—iodo—
propane (l g, 5.649 mmol) was added and the reaction allowed to warm to ambient
temperature over 18 hours. The reaction was quenched by the addition of water and the
e stirred for 10 minutes. The mixture was extracted with diethyl ether (x 3) and the
combined organic extracts washed with water (x 2), brine (x 2), dried (MgSO4), filtered and
concentrated in vacuo to give the sub—title compound that was used directly without r
purification assuming 100% Yield and purity; 1H NMR (500 MHz, DMSO) 5 7.25 — 7.37 (m,
2H) and 7.48 — 7.55 (m, 2H) ppm.
Step 7: 1-Bromo[1,2,2,2-tetradeuterio(trideuteriomethyl)ethyl]sulfonyl—benzene
xxxiii
mCPBA (2.875 g, 12.83 mmol) was added in portions to a stirred solution of 1—
bromo—4—[1,2,2,2—tetradeuterio—1-(trideuteriomethyl)ethyl]sulfanyl—benzene (1.223 g, 5.134
mmol) in DCM (20 mL) at 0 °C and the reaction allowed to warm to ambient temperature
over 17 hours. The mixture was washed 1M aqueous NaOH (X 2), saturated aqueous Na28203
(x 3), brine (x 1), dried (MgSO4), filtered and concentrated in vacuo. The residue was
purified by column tography (ISCO Companion, 80 g column, eluting with 0 to 40%
EtOAc/Petroleum Ether, loaded in DCM) to give the sub—title compound as a colourless oil
(1.19 g, 86% Yield); 1H NMR (500 MHz, DMSO) 8 7.77 - 7.81 (m, 2H) and 7.88 - 7.92 (m,
2H) ppm.
Step 8: 4,4,5,5-Tetramethyl—2-[4-[l,2,2,2-tetradeuterio(trideuteriomethyl)ethyl]
sulfonylphenyl]-1,3,2-dioxaborolane
xxxiv
Pd(dppf)Clz.DCM (179.8 mg, 0.2202 mmol) was added to a stiired suspension
of o—4-[1,2,2,2—tetradeuterio-l~(trideuteriomethyl)ethyl]sulfonyl—benzene (1.19 g,
4.404 mmol), bis(dipinaeolato)diboron (1.342 g, 5.285 mmol) and KOAc (1.296 g, 13.21
mmol) in e (10 mL). The reaction placed under an atmosphere of en via 5 x
nitrogen/vacuum cycles and the mixture was heated at 80 °C for 4.5 hours. The on was
cooled to ambient temperature and the solvent removed in vacuo. The residue was partitioned
between EtZO and water and the layers separated. The organic layer was dried (MgSO4),
PCT/U52012/058127
filtered and concentrated in vacuo. The residue was dissolved in 30% EtOAc/Petroleum ether
(35 mL) and 1.2 g of il was added. The mixture was stirred for 30 minutes then filtered,
washing the solids with further alliquots of 30% EtOAc/Petrol (x 3). The filtrate was
concentrated in vacuo and tritruated from 10% EtOAc/petroleum ether. The resultant solid
was isolated by filtration, washed with petroleum ether and dried in vacuo to give the sub—
title compound as an off—white solid 1 mg, 75% Yield); 1H NMR (400 MHz, DMSO) 8
1.33 (s, 12H), 7.87 (d, J = 8.4 Hz, 2H) and 7.94 (d, J = 8.4 Hz, 2H) ppm.
Step 9: tert—Butyl N—[[4-[5-[3-[bis(tert—but0xycarbonyl)amino]-6—[4—[1,2,2,2—
tetradeuterio-l-(trideuteriomethyl)ethyl]sulfonylphenyl]pyrazin-Z-yl]isoxazol
yllphenyl]methyl]-N—methyl—carbamate
(BOC>2N O’N N\
xxxv
[1, 1’-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(H) (106.8 mg,
0.1639 mmol) was added to a mixture of 4,4,5,5—tetramethy1—2—[4-[1,2,2,2—tetradeuterio—1—
(trideuteriomethyl)ethyl]sulfonylphenyl}l,3,2-dioxaborolane (1.3 g, 4.098 mmol), tert—butyl
N-[[4—[5—[3-[bis(teit-butoxycarbonyl)amino]bromo—pyrazinyl]isoxazol—3-
ny1]methyl]—N—methyl~carbamate (2.707 g, 4.098 mmol) and K2CO3 (1.133 g, 8.200
mmol) in toluene (9.100 mL), EtOH (2.600 mL) and water (2.600 mL) and the reaction
mixture was degassed with a flow of nitrogen (5 cycles).
The mixture was heated at 75 °C for 1.5 hours. The reaction was ccoled to ambient
temperature and water (5.2 mL) was added. After ng the layers were separated and the
organic layer dried (Na2304), d, and concentrated in vacuo. The residue was triturated
with IPA and the ant precipitate isolated by filtration, washed with IPA (3 x 4 mL) and
dried in vacuo at 50 °C to give the title compound as a white solid (2.4 g, 76% Yield); 1H
NMR (400 MHz, CDC13) 6 1.41 (s, 18H), 1.50 (s, 9H), 2.85 — 2.89 (m, 3H), 4.50 (s, 2H),
7.36 ~ 7.38 (m, 3H), 7.87 (d, 2H), 8.09 (d, 2H), 8.35 (d, 2H) and 9.06 (s, 1H) ppm.
WO 49726 PCT/U52012/058127
Step 10: 3-[3—[4-(Methylaminomethy1)phenyl]isoxazol—S-yl][4—[1,2,2,2-tetradeuterio-
1{trideuteriomethyDethyl]sulfonylphenyl]pyrazin—Z-amine (compound 11-4)
NH2 O'N\ HN‘
II-4
Concentrated HCl (3.375 g, 2.812 mL of 37 %w/w, 34.25 mmol) was added to a
solution of tert—butyl N—[[4~[5—[3-[bis(tert~butoxycarbonyl)amino]-6—[4~[l,2,2,2-tetradeuterio(trideuteriomethyl)ethy1]sulfonylphenyl]pyrazin—2—yl]isoxazol—3—yl]phenyl]methy1]-N—
methyl-carbamate (2.2 g, 2.854 mmol) in acetone (28.60 mL) and the reaction heated at
reflux for 7 hours. The reaction was cooled to ambient ature and the resultant
precipitate isolated by filtration, washed with acetone (2 x 4.5 mL) and dried in vacuo at 50
°C to give the di-l-lCl salt of the title compound as a yellow solid ( 1.42 g, 92% Yield); 1H
NMR (400 MHz, DMSO) 5 2.58 (t, 3H), 4.21 (t, 2H), 5.67 (br s, 2H), 7.74 (d, 2H), 7.85 (s,
1H), 7.94 (d, 2H), 8.10 (d, 2H), 8.38 (d, 2H), 8.96 (s, 1H) and 9.33 (br s, 2H) ppm; MS (ES+)
471.8.
PCT/U52012/058127
Exam 1e 6: S nthesis of 5- 4— tert-bu lsulfon l hen l 3- 4-
meth lamino meth l hen lisoxazol-S- l razin-Z-amine Com ound 1-2
HO‘N Step 1 HO‘N Step 2
l I
300 N08, IPAc TEA. DCM
__._____..__, |
\ c|)\©\/BocN\ N(BOC)2
4-i 4-ii N,v\
8%N 4-iii
Step 3 800‘ [Boo 800
N O
Boo [Boo B(OH)2 \
\N \
W0"! \N\
.'POr 23 N] \
8%“l ——-—~—————-——-—>
4-iv 1. cat. Pd(dtbpf)ClZ, PhCHB, 5-i
Br aq. K2C03
2. IPA crystallization
SOQiPI’
NH2 0% HN‘ NH2
4 O’r‘i HN‘
Step
\ Step5 \
\ N \
conc.HCl N! I
acetone / N 4:1 IPA/water
-2HC| / N 'HCI
SOZiPr SOQIPF
Compound I2HC1 Compound I-Z-HCI
Step 1: ation of Compound 4-ii
Hot Step 1 HO\
I I
53°C NCS, IPAc
————-—-——->
N\ cg/K©\/I§ocN\
4-i 4-ii
A suspension of tert—butyl 4~((hydroxyimino)methy1)benzyl (methyl)carbamate
(Compound 4-i) (650 g, 2.46 mol) in isopropyl e (6.5 L) is stirred at ambient
temperature. N—Chlorosuccinimide (361 g, 2.71 mol) is added and the reaction temperature
maintained overnight at 20—28 °C to ensure complete reaction. The reaction mixture is
d with water (3.25 L) and EtOAc (1.3 L) and the phases are separated. The organic
phase is washed with water (2 x 3.25 L), dried (Na2SO4), and trated to a wet-cake. The
concentrate is diluted with e (9.1 L), ~2 L of solvent removed, and then stirred at
ambient temperature for 2—20 h. The solid is collected by filtration. The filter-cake is washed
with heptane (2 x 975 mL) and dried to afford Compound 4-ii (692 g; 94% yield, 99.2 area %
purity by HPLC) as a colorless powder.
Ste 2: Pre aration of ut 1 5—bromo 3— 4- tert—butox carbon 1 meth lamino
meth 1 hen lisoxazol—S- 1 Z— l tert—butox carbon lcarbamate Com ound 4—iv
HO‘N CM B0C\N,Boc
N Boc\
..______, ..
' O\\ N‘
N(Boc)2
Cl 800 // N \
111\ N \ l
l /N
4'"-- Br 4-m 4-Iv_
A suspension of rerr-butyl N—(S-bromo-3 «ethynylpyrazin-Z~yl)~N-tert—
hutoxycarbonylcarbamate (Compound 4-iii)(1.59 kg, 3.99 mol) and tert—butyl 4-
(chloro(hydroxyimino)methyl)benzyl(tetrahydro-2H—pyran—4—y1)carbamate (1.31 kg, 4.39
mol; 1.10 equiv.) in CH2C12 (12.7 L) is stirred at ambient temperature. Triethylamine (444 g,
611 mL, 4.39 mol) is added to the sion and the reaction temperature is maintained
between 20—30 °C for 20—48 h to ensure complete reaction. The reaction mixture is diluted
with water (8 L) and thoroughly mixed, then the phases are separated. The c phase is
washed with water (8 L), dried (NaZSO4), and then concentrated until about 1 L of CH2C12
s. The concentrate is diluted with heptane (3.2 L) and re-concentrated at 40 °C/200 torr
until no distillate is observed. The concentrate is stirred and further diluted with heptane (12.7
L) to precipitate a solid. The suspension is stirred overnight. The solid is collected by
filtration, washed with heptane (2 x 3 L) then dried to afford Compound 4-iv (2.42 kg; 92%
yield, 100 area % purity by HPLC) as a light tan powder. 1H NMR (400 MHz, CDC13) 5 8.61
(s, 1H), 7.82 (d, J= 8.2 Hz, 2H), 7.31 (m, 3H), 4.46 (br s, 2H), 2.84 (br d, 3H), 1.57 (s, 2H),
1.44 (br s, 9H), 1.36 (s, 18H).
PCT/U52012/058127
Ste 3: Pre aration of Com ound 5-i
Boos ,Boc
N B
8(0le o—N\ °°‘N\
Boo Boo
\ / Bog
N OvN \ \
\ N~ iPrOZS NI \
\ /N
| "_““““‘——’
1. cat. pf)012, PhCHg, 5"
4 , IN
Br 3‘1 choa
2. i—PrOH crystallization
SOzlPr
A mixture of tert-butyl (5-bromo—3—(3 —(4—(((tert—
carbonyl)(methyl)amino)methyl)phenyl)isoxazol—S—yl)pyrazin-2~yl)(tert~
butoxycarbony1)carbamate (Compound 4-iv )( 1.00 kg, 1.51 mol), K2CO3 (419 g, 3.02 mol),
and (4—(isopropylsulfonyl)pheny1)boronic acid (345 g, 1.51 mol) in toluene (7.0 L) and water
(2.0 L) was d and degassed with N2 for 30 min. 1,1’—bis(di—t-butylphosphino)ferrocen-
dichloro-palladium(ll) [Pd(dtbpf)C12; 19.7 g, 30.3 mmol] was then added and degassed an
additional 20 min. The on mixture was warmed at 70 °C for at least 1 h to ensure
complete on. The reaction e was cooled to ambient temperature then filtered
through Celite. The reaction vessel and filter pad are rinsed with toluene (2 x 700 mL). The
filtrates are combined and the phases are separated. The organic phase is d with Biotage
MP—TMT resin (170 g) for 4—20 h. The resin is removed by filtration through Celite and the
filter pad is washed with toluene (2 x 700 mL). The filtrate and washings are combined and
concentrated to near dlyness then d with i—PrOH (5.75 L) and re—concentrated. The
concentrate is again dissolved in warm (45 °C) i~PrOH (5.75 L) and then cooled to ambient
temperature with stirring to induce crystallization then stirred for around 16 — 20 h. The solid
is collected by filtration, washed with i—PrOH (2 x 1 L), and dried to afford VRT—1018729
(967 g; 84%) as a beige powder. 1H NMR (400 MHz, CDC13) 6 9.04 (s, 1H), 8.33 (d, J = 8.6
Hz, 2H), 8.06 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.1 Hz, 2H), 7.34 (m, 3H), 4.47 (br s, 2H),
3.25 (hept, J= 7.0 Hz, 1H), 2.85 (br d, 3H), 1.47 (s, 9H), 1.38 (s, 18H), 1.33 (d, J= 6.9 Hz,
6H).
Step 4: Preparation of Compound 1-2 ' 2HCl
A solution of Compound S-i (950 g, 1.24 mol) in acetone (12.35 L) is warmed to
40 °C then concentrated HCl (1.23 kg, 1.02 L of 37 %w/w, 12.4 mol) is added at a rate to
maintain the reaction temperature between 40 - 45 °C for at least 5 h to ensure complete
reaction. The suspension is cooled to below 30 °C and the solid collected by filtration. The
2012/058127
cake is washed with acetone (2 x 950.0 mL) then dried to afford Compound I—2 ' 2HC1
(578 g; 87% yield, 99.5 area % purity by HPLC) as a yellow powder. 1H NMR (400 MHz,
DMSO) 6 9.53 (br d, J= 4.8 Hz, 2H), 8.93 (s, 1H), 8.37 (d, J= 8.5 Hz, 2H), 8.07 (d, J= 8.3
Hz, 2H), 7.92 (d, J = 8.6 Hz, 2H), 7.84 (s, 1H), 7.75 (d, J = 8.3 Hz, 2H), 4.23 — 4.15 (m, 2H),
3.43 (hept, J= 6.8 Hz, 1H), 2.55 (t, J= 5.3 Hz, 3H), 1.17 (d, J= 6.8 Hz, 6H).
Ste 5: Pre aration of Com ound I-2 - HCI from Corn ound 1-2 - 2HC1
NH2 O‘N\ HN‘ NH2 O’N\ HN\
\ \
N \ N \
I . |
/ N 4.1 I-PrOH/water / N -HCl
-2HCI
SOZiPI' SOQiPr
Compound l-2 0 2HC| Compound l-2 - HCI
Two-pot process
] A stirred suspension of Compound I-2 ' 2HC1 (874 g, 1.63 mol) in i-PrOH (3.50
L) and water (0.87 L) is warmed at 50 °C for 1—2 h, cooled to ambient temperature, and
stirred for 1-20 h. XRPD is performed on a small sample to ensure that Compound 1—2 °
2HC1 has been converted to another form. The suspension is cooled to 5 °C and stirred for 1
h. The solid is collected by filtration then the filter-cake is washed with 80/20 i-PrOH/water
(2 x 874 mL), and briefly dried.
IfXRPD shows the Compound 1—2 - HCI/anhydrate form, the solid is dried to
afford nd 1—2 °HCl/anhydrate (836 g, 99% yield, 99.2 area % purity by HPLC) as a
yellow solid. 1H NMR (400 MHZ, DMSO) 5 9.38 (s, 2H), 8.96 (s, 1H), 8.46 ~ 8.34 (m, 2H),
8.10 (d, J= 8.3 Hz, 2H), 7.94 (d, J= 8.6 Hz, 2H), 7.85 (s, 1H), 7.75 (d, J= 8.3 Hz, 2H), 7.23
(br s, 2H), 4.21 (3, 21-1), 3.47 (hept, J = 6.7 Hz. 11-1), 2.58 (s, 3H), 1.19 (d, J= 6.8 Hz, 6H).
IfXRPD shows the Compound 1-2 -HCl/hydrate form the solid is stirred in
fresh i-PrOH (3.50 L) and water (0.87 L) at 50 °C for at least 2 h until XRPD shows complete
conversion to Compound 1—2 'HCI/anhydrate. The suspension is then cooled to 5 °C and
stirred for 1 h. The solid is collected by filtration then the cake is washed with 80/20 i-
PrOH/water (2 x 874 mL) then dried to afford Compound 1HC1/anhydrate.
Alternative procedure (single pot) used
Compound 1-2 0 2HC1 (392 g) is charged to the reactor. 4:] IPA/water (8 L) is
charged to a reactor and stirred at ambient temperature overnight. XRPD is used to confirm
the conversion to the mono-HCI salt mono—hydrate form. The mixture is heated to 50 °C.
Seeds of nd 1-2 ' HCI/anhydrate (16 g) are added and the e heated at 50 °C
until XRPD confirms complete conversion to the desired anhydrate form. The mixture is to
cooled to ambient, filtered and the solid washed with 4:1 IPA/water (2 x 800 mL) then dried
to afford Compound 1—2 - HCI/anhydrate (343 g, 94% yield).
Ste 4: Alternate Method 1: Pre aration of Com ound I-2 free base
fl;W 1. TFA N/Me DCM
HNrMe
25 c'C
- 2 NaOH 0~N
tPrOZS
EtOH/water iPrOZS
nd 1-2 (free base)
A solution of Compound 5-i (100 g, 131 mmol) in DCM (200 mL) was stirred
at ambient temperature then TFA (299 g, 202 mL, 2.62 mol) was added. After 2 h reaction
solution was cooled to 5 °C. The reaction e was diluted with EtOH (1.00 L) over about
min resulting in a bright yellow suspension. The suspension was cooled to 10 °C then
NaOH (1.64 L of 2.0 M, 3.28 mol) was added over 30 min then stirred at ambient
temperature overnight. The solid was ted by filtration then washed with water (2 x 400
mL), EtOH (2 x 200 mL) then dried to afford Compound 1—2 free—base (57.0 g, 94% yield,
99.7 area % purity by HPLC) as a fine, yellow powder. 1H NMR (400 MHz, DMSO) 5 8.95
(s, 1H), 8.39 (d, J= 8.5 Hz, 2H), 7.95 (dd, J: 11.6, 8.4 Hz, 4H), 7.78 (s, 1H), 7.51 (d, J= 8.2
112,211), 7.21 (br s, 2H), 3.72 (s, 2H), 3.47 (hept, J= 6.8 Hz, 1H), 2.29 (s, 3H), 1.19 (d, J=
6.8 Hz, 6H).
Ste 4: Alternate Method 2: Pre aration of Com ound 1-2 - HCI
N NHZ
/ 'HCI
’Me [M
N /
Acetone OxN
iPrOZS 1P1’028
nd I-2 Compound I-2 HCI
W0 2013I049726 PCT/U82012/058127
A suspension of Compound I—2 free base (10.0 g, 21.6 mmol) in acetone (80
mL) was stirred and heated to 35 °C. An s on of HCl (11.9 mL of 2.0 M, 23.8
mmol) diluted with water (8.0 mL) was added and the e heated at 50 °C for 4 h. The
suspension was allowed to cool to ambient temperature then stirred overnight. The solid was
collected by filtration. The filter—cake was washed with acetone (2 x 20 mL) then dried to
afford 10.2 g Compound 1-2 hydrochloride (95% yield) as a yellow powder.
[002051 Exam 1e 7: S nthesis of 5- 4- Iso ro lsulfon l hen l 3- 4-
tetrah dro ran~4- lamina meth 1 hen l isoxazol—S- l razin—Z-amine Com ound
Scheme: Exam le S nthesis of Com ound 1-3
B(OH)2
oC>——NBoc OC>7N,Boc N(Boc)}
/N SOQi-Pr
A—5-i
A4 "
NCS, i-PrOAc Br *—'~’
_.__..__>
, 1. cat Pd(dtbpf)C|2,
,N~ 20 — 30 °c ,N— TEA, DCM PhCHg, aq. K2003
HO HO Cl 20 - 30 °C 2. EtOH llization
3. MP-TMT resin
A-4 Ai A.5
1. TFA NHZ
I \N
DCM ,
- 25 °C
__.. N, \
2. NaOH
EtOH/water
SOzi—Pr 502“Pr
A-6 L3
PCT/U$2012/058127
EtN(i—Pr)2, NaBH4
—> ti?)
MeOH
NH2'HCI 20 - 25 °C
A on of tetrahydro-2H—pyran-4—amine hydrochloride (1.13 kg, 8.21 mol)
in MeOH (14.3 L) is d at about 20 °C. then Et3N (1.06 kg, 1.43 L, 8.21 mol) is added.
The mixture is stirred for at least 5 min then terephthalaldehyde diethyl acetal (1.43 kg, 6.84
mol) is added while maintaining the reaction temperature between 20—25 °C. The mixture is
stirred for at least 45 min to form the imine. NaBH4 caplets (414 g, 11.0 mol) are added while
maintaining the reaction temperature below about 25 °C. The mixture is stirred for 1 h after
the addition is completed. The reaction mixture is quenched by adding 1 M NaOH (13.7 L)
then extracted with MTBE. The organic solution was washed with brine (7.13 L) then dried
(Na2SO4) and concentrated to afford Compound A—2 (2197 g; 109% yield, 94.4 area % purity
by HPLC) as a hazy oil. 1H NMR (400 MHz, CDC13) 8 7.43 (d, J= 8.1 Hz, 2H), 7.31 (d, J=
8.1 Hz, 2H), 5.49 (s, 1H), 4.66 (br s, 1H), 4.03 — 3.91 (m, 2H), 3.82 (s, 2H), 3.69 — 3.47 (m,
4H), 3.38 (td, J= 11.6, 2.1 Hz, 2H), 2.78 — 2.65 (m, 1H), 1.90— 1.81 (m, 2H), 1.53 — 1.37 (m,
2H), 1.23 (t, J = 7.1 Hz, 6H).
Ste 2: Pre aration of tert-but l4- diethox meth lben ltetrah dro-ZH- ran-4—
yllcarbamate gA-32
O Boc 0, Et N
NH DCM
— 25 °C tMQOC
A mixture of N—(4-(diethoxymethyl)benzyl)tetrahydro-ZH—pyran-4—amine (A-2)
(2195 g, 7.48 mol) in CH2C12 (22.0 L) is stirred at 25 °C then di—t-butyl dicarbonate (1.71 kg,
7.86 mol) is added. RN (795 g, 1.10 L) is then added while ining the reaction
temperature between 20 — 25 °C. The reaction mixture is d at about 25 °C for 12 — 20 h.
After the reaction is completed, the mixture is cooled to about 20 °C and quenched with 0.5
M aqueous citric acid (7.48 L, 3.74 mol) while maintaining the reaction temperature between
W0 20131049726 PCT/U52012/058127
— 25 °C. The c phase is collected, washed with sat. NaHC03 (6.51 L, 7.48 mol),
washed with brine (6.59 L), and dried (N212804) then concentrated to afford tert-butyl 4-
(diethoxymethyl)benzy1(tetrahydro-2H-pyran-4—yl)carbamate (A-3) (2801 g; 95% yield, 98.8
area % purity by HPLC) as a thick, amber oil.1H NMR (400 MHZ, CDC13) 6 7.40 (d, J= 8.1
Hz, 2H), 7.21 (d, J= 7.9 Hz, 2H), 5.49 (s, 1H), 4.39 (br s, 3H), 3.93 (br dd, J: 10.8, 3.8 Hz,
2H), 3.67 — 3.47 (m, 4H), 3.40 (br m, 2H), 1.68 - 1.59 (m, 4H), 1.39 (br s, 9H), 1.23 (t, J=
7.1 Hz, 6H).
Ste 3: Pre aration of tert-bu l4-
4—yl)carbamate gA-4)
0 0
OEt HOT"
Etc/‘\[>V NH20H'HCI
——————-—->
N‘Boc K©V
THF/HZO N‘Boc
- 25 ‘JC
A-3 A-4
A solution of tert—butyl 4—(diethoxymethy1)benzyl(tetrahydro—2H—pyran—4—
yl)carbamate (A-3) (2.80 kg, 7.12 mol) in THF (28.0 L) and water (2.80 L) is d at about
°C. Hydroxylamine hydrochloride (593 g, 8.54 mol) is added while ining the
reaction ature between 20—25 °C. The reaction mixture is stirred at about 20 °C for 16
— 20 h then diluted with CHzClz (8.4 L) and 50% brine (11.2 L) and stirred for at least 5 min.
The phases are separated then the organic phase is washed with 50% brine (2 x 2.8 L), dried
(Na2SO4) and concentrated. The concentrate is diluted with MeOI—I (1.4 L) and re—
concentrated. The concentrate is diluted with MeOH (14.0 L) and transferred to a reaction
vessel. The solution is warmed to about 25 °C then water ( 14.0 L) is added over about 1 — 1.5
h; after about 10 L of water is added, the mixture is seeded and a hazy suspension is
observed. Additional water (8.4 L) is added over 1.5 h to further precipitate the product. After
aging, the solid is ted by filtration. The filter-cake is washed with heptane (5.6 L) and
dried to afford tert—butyl 4-((hydroxyimin0)methyl)benzy1(tetrahydro—2H-pyran
yl)carbamate (A—4) (1678 g; 71%, 91.5 area % purity by HPLC) as an off—white powder. 1H
NMR (400 MHz, CDCl3) 6 8.12 (s, 1H), 7.51 (d, J= 8.2 Hz, 2H), 7.24 (d, .I= 7.9 Hz, 2H),
4.40 (br s, 3H), 3.96 (dd, J= 10.4, 3.6 Hz, 2H), 3.41 (br m, 2H), 1.69 — 1.61 (m, 4H), 1.39 (br
s, 9H).
PCT/U52012/058127
Ste 4: Pre n of tart—bu 14— chloro h drox imino meth lbenz l tetrah dro-
2H- ran lcarbamate Ai
HO\ O
B00 20 30 °C
A-4 i
A suspension of (E)-z‘erIf—buty1 4—((hydroxyimino)methy1)benzyl(tetrahydro—2H—
4—y1)carbamate (A4) (1662 g, 4.97 mol) in i—PrOAc (16.6 L) is stirred at 20 °C in a
reactor. N—chlorosuccinimide (730 g, 5.47 mol) is added maintaining about 20 °C. The
suspension is stirred at about 20 °C to complete the reaction. The suspension is diluted with
water (8.3 L) and stirred to dissolve the solid. The phases are separated and the organic phase
is washed with water (8.3 L). The organic phase is concentrated then diluted with c
(831 mL). e (13.3 L; 8 V) is slowly added to induce crystallization. The thick
suspension is then stirred for 1 h. The solid is collected by filtration; the filter—cake is washed
with heptane (2 x 1.6 L; 2 x 1 V) and dried to afford (Z)—tert—butyl 4-
(chloro(hydroxyimino)methy1)benzy1 (tetrahydro-2H-pyranyl)carbamate (A—4-i) (1628 g;
89%, 98.0 area % purity by HPLC) as a white .
Ste 5: Pre aration of (err-but l 5-bromo 3- 4- tert-butox carbon 1 tetrah dro-
ZH- ran-4— lamino meth l hen lisoxazol-S- l razin-Z- l tert-
butox carbon lcarbamate A-5
TEA, DCM Boc\ /
QKKNZAii \
NBC N \
Br 00
Al
A solution of terl-butyl 4-(ch1oro(hydroxyimino)methy1)benzy1(tetrahydro-2H-
pyrany1)carbamate (Ai) (1.60 kg, 4.34 mol) and rert-butyl N—(S—bromo
ethynylpyrazin-2—yl)—N—tert—butoxycarbonylcarbamate und A—4-ii) (1.73 kg, 4.34
mol) in CH2C12 (12.8 L) is stirred at 20 °C. Et3N (483 g, 665 mL; 4.77 mol) is added and the
reaction temperature maintained below 30 °C. The suspension stirred at 20 °C to complete
PCT/U52012/058127
the reaction then diluted with water (8.0 L) and agitated. The phases are separated and the
c phase is washed with water (8.0 L) and then concentrated. i-PrOAc (1.6 L) is added
and the mixture and heated at 50 °C. Heptane (4.0 L) was slowly added then the suspension is
allowed to cool to ambient temperature and stirred overnight. Additional heptane (7.2 L) is
added to the suspension and it is d for l h. The solid is collected by filtration. The filter-
cake is washed with hcptane (2 x 1.6 L) and dried to afford tert—butyl (5-bromo—3 —(3—(4—
(((tert-butoxycarbony1)(tetrahydro—2H-pyran-4—yl)amino)methyl)phenyl)isoxazol—5—
azin—2—yl)(tert—butoxycarbonyl)carbamate (A~5) (2.478 kg; 78%, 97.8 area % purity by
HPLC) as a fine, tan powder. 1H NMR (400 MHz, CDC13) 5 8.60 (s, 1H), 7.78 (d, J= 8.3 Hz,
2H), 7.31 (m, 3H), 4.42 (br m, 3H), 4.03 — 3.82 (m, 2H), 3.38 (br s, 2H), 1.60 (m, 4H), 1.36
(s, 27H).
Ste 6: Pre aration of tert-bu ltert-butox carbon 13- 3— 4— tert-
butox carbon 1 tetrah dro-ZH- ran lamino meth l hen lisoxazol-S- 1 4-
iso r0 lsulfon l hen 1 razin-Z— lcarbamate
i-PrOzS NWl N‘Boc
N \ Ai / N
l N\BOC ..........___..—>
’ N (j 1. cat. Pd(dtbpf)C|2, PhCHg. o
aq. K2C03
2. EtOH crystallization
A-5 A's
3. MP-TMT resin sozi'Pr
A mixture of terl—butyl (5-bromo~3—(3 —(4—(((tert-butoxycarbonyl)(tetrahydro—
an-4~yl)amino)methyl)phenyl)isoxazol—5—yl)pyrazin-2—yl)(zert-
butoxycarbonyl)carbamatc (A-S) (425 g, 582 mmol), K2C03 (161 g, 1.16 mol; 2.0 equiv.),
and (4-(isopropylsulfonyl)phenyl)boronic acid (133 g, 582 mmol) in toluene (2.98 L) and
water (850 mL) is stirred and degassed with N2 at ambient temperature. The catalyst [1,1’-
-Zert—butylphosphino)ferrocenc]dichloropalladium(ll), (Pd(dtbpf)Clz; 1.90 g, 2.91
mmol) is added and the e is degassed for an additional 10 min. The mixture is heated at
70 °C until the reaction is complete. The e is cooled to 50 °C, diluted with water (850
mL) and filtered through a bed of Celite. The phases are separated. The organic phase is
concentrated then the residue is diluted with EtOH (1.70 L) and rc-conccntrated. With mixing
at 40 °C, the trate is diluted with EtOl—l (1.70 L) to induce crystallization. The
W0 20131049726
suspension is cooled to 20 °C and stirred for 4 h. The solid is collected by filtration. The
filter-cake is washed with EtOH (2 x 425 mL) and air-dried to afford tert-butyl tert-
butoxycarbonyl(3-(3 -(4-(((tert—butoxycarbonyl)(tetrahydro-2H—pyran-4—
yl)amino)methyl)phenyl)isoxazol—S—yl)—5—(4—(isopropylsulfonyl)pheny1)pyrazin
y1)carbamate (A—6) as a beige powder. The solid is ved in THF (2.13 L) and slurried
with Biotage MP—TMT resin (48 g) at ambient temperature. The resin is removed by filtration
and the filtrate concentrated to remove most of the THF. The concentrate is d with
EtOH (970 mL) and re—concentrated to about half the original volume. The concentrate is
diluted again with EtOH (970 mL) and mixed for 1 h at 40 °C. The suspension is cooled to
ambient temperature and the solid is ted by filtration then dried to afford tert—butyl tert—
butoxycarbonyl(3—(3 —(4—(((tert-butoxycarbonyl)(tetrahydro—ZH—pyran—4-
y1)amino)methyl)phenyl)isoxazol~5-yl)-5—(4—(isopropy1sulfonyl)pheny1)pyrazin
yl)carbamate (A—6) (416 g; 86% yield, 99.3 area % purity by HPLC) as a white powder. 1H
NMR (400 MHz, CDC13) 5 9.04 (s, 1H), 8.38 — 8.28 (m, 2H), 8.10 — 8.01 (m, 2H), 7.82 (d, J
= 8.2 Hz, 2H), 7.34 (m, 3H), 4.44 (br s, 2H), 3.94 (dd, J= 10.5, 3.5 Hz, 2H), 3.40 (br s, 2H),
3.25 (hept, .]= 6.8 Hz, 1H), 1.65 (m, 4H), 1.38 (br s, 27H), 1.33 (d, .]= 6.9 Hz, 6H).
Ste 7: Pre aration of 5- 4- ’
4- lamina meth l hen lisoxazol—S- l razin-Z-amine 1-3 freebase form
BOC‘N’BO(Z)"‘1\NW NH2 0—11
1. TFA NW
I N‘Boc DCM I NH
/ N (j N
— 25 °c /
———-——>‘
o 2. NaOH (:30
ater
A-6 [-3
SOziPr SOziPr
A sion of tert—butyl tert—butoxycarbonyl(3—(3—(4-(((tert—
butoxycarbonyl)(tetrahydro-2H-pyranyl)amino)methyl)phenyl)isoxazol-5—yl)-5—(4-
(isopropylsulfony1)phenyl)pyrazin—2-y1)carbamate (A~6) (410 g; 492 mmol) in CH2C12 (410
mL) is d at ambient temperature in a flask. TFA (841 g, 568 mL; 7.4 mol) is added
while maintaining the reaction temperature n 20—25 °C. The solution is stirred at
ambient temperature for about 3 h when analysis shows reaction completion. The solution is
cooled to about 5—10 °C and diluted with EtOH (3.3 L) while maintaining the temperature
below 20 °C. A 5.0 M aqueous solution ofNaOH (1.77 L; 8.85 mol) is added while allowing
the reaction temperature to rise from about 14°C to about 42 °C. The suspension is heated at
70 — 75 °C for 6 h while removing distillate. The sion is allowed to cool to ambient
temperature. The solid is collected by filtration and the filter-cake is washed with water (4 x
1.64 L). The filter—cake is washed with EtOH (2 x 820 mL) and dried to afford 5—(4—
(isopropylsulfonyl)phenyl)—3~(3—(4—(((tetrahydro—ZH—pyrany1)amino)methyl)phenyl)
ol-S-yl)pyrazin—2—amine (Compound 1—1) (257 g; 98% yield, 99.5 area % purity by
HPLC) as a yellow powder.
1H NMR (400 MHz, DMSO) 6 8.94 (s, 1H), 8.44 - 8.33 (m, 2H), 7.94 (t, J=
8.2 Hz, 4H), 7.76 (s, 1H), 7.53 (d, J= 8.2 Hz, 2H), 7.20 (s, 2H), 3.83 (m, 1H), 3.80 (s, 3H),
3.46 (hept, J= 6.8 Hz, 1H), 3.25 (td, J= 11.4, 2.1 Hz, 2H), 2.66 — 2.54 (m, 1H), 1.79 (br dd,
2H), 1.36 — 1.22 (m, 2H), 1.19 (d, J= 6.8 Hz, 6H).13C NMR (101 MHz, DMSO) 5 167.57,
151.76, 141.07, 137.58, 135.75, 129.16, 128.53, 126.57, 126.41, 125.69, 124.52, 102.13,
65.83, 54.22, 52.60, 49.19, 33.18, 15.20.
Compound ical Data
Cmpd LCMS LCMS
HNMR
N0. ES + Rt min
H NMR (400 MHz, DMSO) 6 9.63 (d, J = 4.7 Hz, 2H),
9.05 (s, 1H), 8.69 (d, J= 5.2 Hz, 1H), 8.21 (s, 1H), 8.16 —
8.03 (m, 3H), 7.84 (t, J= 4.1 Hz, 3H), 7.34 (br s, 2H), 4.40
— 4.18 (m, 2H), 3.94 (dd, J=11.2, 3.9 Hz, 2H), 3.32 (t, J=
11.2 Hz, 3H), 2.17 — 2.00 (m, 2H), 1.81 (s, 6H), 1.75 (dd, J
12.1, 4.3 Hz, 2H.
H NMR (400 MHz, DMSO) 5 8.94 (s, 1H), 8.44 - 8.33 (m,
2H), 7.94 (t, J= 8.2 Hz, 4H), 7.76 (s, 1H), 7.53 (d, J= 8.2
Hz, 2H), 7.20 (s, 2H), 3.83 (m, 1H), 3.80 (s, 3H), 3.46
(hept, J= 6.8 Hz, 1H), 3.25 (td, J= 11.4, 2.1 Hz, 2H), 2.66
— 1.22 (m, 2H), 1.19
— 2.54 (m, 1H), 1.79 (br dd, 2H), 1.36
(d, J= 6.8 Hz, 6H).
1H NMR (500 MHz, DMSO) 9.10 (d, J = 5.8 Hz, 2H), 8.97
(s, 1H), 8.42 — 8.37 (m, 2H), 8.15 - 8.08 (m, 2H), 7.99 — 7.92
II—l 466.2 0.83 (m, 2H), 7.85 (s, 1H), 7.75 — 7.69 (m, 2H), 7.22 (br s, 2H),
3.48 (hept, J = 6.8 Hz, 1H), 2.60 (t, J = 5.4 Hz, 3H), 1.20 (d,
J = 6.8 Hz, 6H).
1H NMR (500 MHz, DMSO) 9.14 (s, 2H), 8.96 (s, 1H),
8.42 ~ 8.36 (m, 2H), 8.13 - 8.08 (m, 2H), 7.99 — 7.91 (m,
II—2 469.1 0.82
2H), 7.85 (s, 1H), 7.78 - 7.68 (m, 2H), 7.21 (s, 2H), 3.48
(d J= 13.6, 6.7 Hz, 1H 1.20
, , d, J= 6.8 Hz, 6H).
WO 2013049726 PCT/U52012/058127
1H NMR (500 MHz, DMSO) 9.11 (s, 2H), 8.97 (s, 1H),
8.39 (d, J = 8.7 Hz, 2H), 8.11 (d, J = 8.4 Hz, 2H), 7.95 (d, J
= 8.7 Hz, 2H), 7.85 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.23
(s, 2H), 4.25 - 4.19 (m, 2H), 3.47 (tt, J = 14.0, 6.9 Hz, 1H),
1.20 (d, J = 6.8 Hz, 6H).
1H NMR (400 MHz, DMSO) 5 2.58 (t, 3H), 4.21 (t, 2H),
.67 (br s, 2H), 7.74 (d, 2H), 7.85 (s, 1H), 7.94 (d, 2H), 8.10
(d, 2H), 8.38 (d, 2H), 8.96 (s, 1H) and 9.33 tbr s, 2H} ppm
INTERMEDIATES
Example 8: Preparation of Oxime 5a
SCHEME BB:
Step 1b OEt Step 2b
——-————>
NaBH4 N\ DCM
OEt HO‘
Et0)\©\/§OC Step 3b 1“
NHZOH'HCI 1300
N _...____., I
\ N
THF/water \
3b 53
Step 1b
Add MeOH (28.00 L) and 4—(diethoxymethyl)benzaldehyde (Compound 1b)
(3500 g, 16.81 mol) into a reactor at 20 °C. Add amine, 33% in EtOH (1.898 kg, 2.511
L of 33 %w/w, 20.17 mol) maintaining 20-30 °C then stir for 1.5 h to form the iminc. Add
NaBH4 (381.7 g, 10.09 mol)cap1ets maintaining the temperature between 20 - 30 °C. Stir at
room temperature for at least 30 min to ensure complete reaction. Add s NaOH (16.81
L of 2.0 M, 33.62 mol) maintaining approximately 20 °C. Add MTBE (17.50 L) and brine
(7.0 L), stir for at least 5 min then allow the phases to te. Extract the aqueous layer
with MTBE (7.0 L) then combine the organic phases and wash with brine (3.5 L) then dry
04) then concentrate to 6 L. The biphasic mixture was transferred to a separatory funnel
and the aqueous phase removed. The organic phase was concentrated to afford 1-(4-
(diethoxymethyl)phenyl)-N-methylmethanamine (Compound 2b) (3755 g, 16.82 mol, 100%
WO 49726 ‘ PCT/U82012/058127
yield) as an oil. ‘H NMR (400 MHz, CDClg) 5 7.43 (d, J= 8.1 Hz, 2H), 7.31 (d, J: 8.1 Hz,
2H), 5.49 (s, 1H), 3.75 (s, 2H), 3.68 — 3.46 (m, 4H), 2.45 (s, 3H), 1.23 (t, J= 7.1 Hz, 6H).
Steps 2b and 3b
Add F (15.00 L) and l—(4-(diethoxymethyl)phenyl)—N—
methanamine (Compound 2b) (3750 g, 16.79 mol) to a r at 20 °C. Add a
solution of B00 anhydride (3.848 kg, 4.051 L, 17.63 mol) in 2-MeTHF (7.500 L) maintaining
approximately 25 °C. Stir for at least 30 min to ensure complete conversion to tert—butyl 4-
(diethoxymethyl)benzyl(methyl)carbamate (Compound 3b), then add a solution ofNaZSO4 _
(1.192 kg, 8.395 mol) in water (11.25 L). Heat to 35 °C then add a solution of hydroxylamine
hydrochloride (1.750 kg, 25.18 mol) in water (3 .75 L) then stir for at least 6 h to ensure
complete reaction. Cool to 20 °C, stop the stirring and remove the aqueous phase. Wash the
c layer with brine (3.75 L), dry (Na2SO4), filter and concentrate to about 9 L. Add
heptane (15.00 L) and crystalline utyl 4—((hydroxyimino)methyl)benzy1(methyl)
carbamate (Compound 5a) (1.0g portions every 10 min) until nucleation was evident, then
concentrate to afford a solid slurry. Add heptane (3.75 L) then cool to room temp and filter.
Wash with heptane (5.625 L) then dry to afford tert—butyl 4—((hydroxyimino)methyl)
benzyl(methyl)carbamate (Compound 5a) (4023 g, 15.22 mo], 91 % yield, 972 area % purity
by HPLC) as a colorless solid. 1H NMR (400 MHz, CDC13) 5 8.13 (s, 1H), 7.54 (d, J= 8.1
Hz, 2H), 7.25 (br (1, 2H), 4.44 (br s, 2H), 2.83 (br d, 3H), 1.47 (br s, 9H).
Scheme CC: Synthesis of Intermediate Aii
NH2 NH2 TMS
N)\rBr /
' l
, N 'PTSA
Sonogashira BOC protection
Bf --——-——-———-———> Br —--——->
C-1 C-2
N B TMS N Boc
N \ N \
RN TMS removal
/ RN/
Br Br
C-3 Aii
The compound of formula A-4—ii may be made according to the steps outlined
in Scheme C. Sonogashira coupling reactions are known in the art (see e.g., Chem. Rev.
WO 2013049726 PCT/U82012/058127
2007, 874-922). In some embodiments, suitable Sonogashira coupling conditions se
adding 1 equivalent of the compound of formula C-1, 1 equivalent of TMS-acetylene, 0.010
lents of Pd(PPh3)2C12, 0.015 equivalents of CuI and 1.20 equivalents ofNMM in
isopropanol. The product can be ed by adding water to the alcoholic on mixture.
Amine salts of a product maybe formed by dissolving the amine in a common,
c solvent and adding an acid. Examples of le solvents include chlorinated
solvents (e. g., dichloromethane (DCM), dichloroethane (DCE), CHzClz, and chloroform),
ethers (e.g., THF, 2—MeTI-IF and dioxane), esters (e.g., EtOAc, IPAC) and other aprotic
solvents. Examples of suitable acids include but are not limited to HCl, H3PO4, H2304, MSA,
and PTSA. In some embodiments, the solvent is IPAC and the acid is PTSA. In some
embodiments, the acid addition salt is converted back to the free amine base in the presence
of a suitable solvent and a le base. le solvents include EtOAc, IPAC,
dichloromethane (DCM), dichloroethane (DCE), CH2C12, chloroform, 2—MeTHF, and
suitable bases include NaOH, NaHCO3, Na2C03, KOH, KHCOg, K2C03, and C52CO3. In
some embodiments, the suitable solvent is EtOAc and the suitable base is KHCO3,
The amine of Compound 02 may be protected with s amine ting
groups, such as Boc (tert-butoxycarbonyl). Introduction of Boc protecting groups is known in
the art (see e.g. Protecting Groups in Organic Synthesis, Greene and Wuts). In some
ments, suitable ions involve adding 1.00 equivalents of the amine, 2.10
equivalents of di—tert—butyl dicarbonate, and 0.03 equivalents ofDMAP in EtOAc.
Reduction in Pd is achieved by treating with a metal scavenger a gel,
functionalized resins, charcoal). In some embodiments, suitable conditions involve adding
charcoal.
The TMS (trimethylsilyl) protecting group on Compound C-3 may be removed
via conditions known to one of skill in the art. In some embodiments, TMS removal
conditions comprise reacting the TMS-protected compound with a suitable base in a suitable
solvent. Examples of suitable solvents include chlorinated solvents (e.g., dichloromethane
(DCM), dichloroethane (DCE), CH2C12, and chloroform), ethers (e.g., THF, 2—MeTHF and
dioxane), esters (e.g., EtOAc, IPAC), other aprotic solvents and alcohol solvents (e. g.,
MeOH, EtOH, iPrOI—I). Examples of suitable bases include but are not limited to (e.g., NaOH,
KOH, K2CO3, Na2C03). In certain ments, suitable conditions comprise adding 1.00
equivalents ofthe TMS-protected acetylene, 1.10 equivalents of K2CO3, EtOAc and EtOH. In
some emboments, the alcoholic solvent, such as EtOH, is added last in the reaction. In some
embodiments the t acetylene is isolated by adding water.
PCT/U82012/058127
Scheme DD: Example Synthesis of Compound Aii
NH2 1. TMS4acetylene NH2 TMS
cat. Pd(PPh3)ZCI2
Br ¢ 1. EtOAc, aq. KHCOS
N \ cat. Cul, NMM, IPA N \ 2. Boch, cat. DMAP,
RN 2. water ”\fN °PTSA EtOAc
3. PTSA, EtOAc 3. charcoal
Br —-——————> Br ———"‘“>
C4 (65-75%) 0.2 (95-10005)
N(Boc)}/ TMS 1. K2003, EtOAc,
EtOH
2. water NV”(800);
krN/ ————-> K/N/
Br (75-80%) Br
C-3 Aii
Exam 1e 9: S nthesis of Com ound Aii
NIV/
A—4-ii
Ste 1: Pre aration of 5-bromo trimeth lsil leth n l razin-Z-amine Com ound
9.2.1
NH2 1. TMS—acetylene NH2 TMS
cat. 3)2C|2
Br é
NI \ cat. Cul, NMM, [PA N \
RN 2. water “\(N 'PTSA
3. PTSA, EtOAc
Br —.__——.———.> Br
c-1 (65—75%) c-2
] Charge isopropanol (8.0 L) to a reactor the stir and sparge with a stream of N2.
Add 3,5—dibromopyrazinamine (Compound GD (2000 g, 7.91 moles), Pd(PP'h3)2C12 (56 g,
0.079 moles), CuI (23 g, 0.119 moles), and NMM (1043 mL, 9.49 moles) to the reactor under
a N2 atmosphere. Adjust the on ature to 25 °C. Purge the reactor with N2 by
doing at least three vacuum/N2 purge cycles. Charge ”EMS—acetylene (1.12 L, 7.91 moles) to
the reaction mixture and maintain the reaction temperature below 30 °C. When the reaction is
complete lower the temperature of the reaction mixture to 15 °C then add water (10 L) and
PCTfUS2012/058127
stir for at least 2 h. The solid is collected by filtration washing the solid with 1:1 IPA/water
(2 x 6 L). The filter cake is dried under vacuum then charged to a reactor and dissolved in
EtOAc (12.5 L). PTSA e (1.28 kg, 6.72 mol) is charged as a solid to the reactor. The
mixture is stirred at ambient temperature for at least 5 h then the solid is collected by
filtration, washed with 1:1 heptane/EtOAc (3.5 L) ed by heptane (3.5 L). The filter
cake is dried to afford 5—bromo-3 —((trimethylsily1)ethynyl)pyrazin-2—aminc(Compound 02)
as a PTSA salt (2356 g, 67% yield, 98.9 area % purity by HPLC).1H NMR (400 MHZ,
DMSO) 8 8.12 (s, 1H), 7.48 (d, J= 8.1 Hz, 2H), 7.12 (d, J: 8.0 Hz, 2H), 2.29 (s, 3H), 0.26
(s, 9H).
Steps 2 and 3
1. EtOAc, aq. KHC03
2. 80020. cat. DMAP.
NH2 TMS 1. KZCOS EtOAc
é EtOAc N(BOC)} TMS
EtOH ”(300);
N \ 3.charcoa|
NV 2.water /
l \
/N oPTSA W9 |
0%) N
(75-80%) /
Br Br
c.3 Aii
Ste 2: Pre aration of u lN-tert-butox carbon l—N— 5-brom0
eth n l razin-Z- lcarbamate Com ound C-3
A solution of5-brom0—3—((trimethylsilyl)ethynyl)pyrazin—2—amine PTSA salt
(Compound C-2) (2350 g, 5.31 mol) in EtOAc (11.5 L) is stirred with a 20% w/w aq. on
of KHC03 (4.5 kg, 1.5 eq.) for at least 30 min. The layers are separated and the c layer
is concentrated then dissolved in EtOAc (7 L) and added to a reactor. DMAP (19.5 g, 0.16
mol) is added followed a solution of Boczo (2436 g, 11.16 mol) in EtOAc (3 L) is added
lowly. The reaction is stirred for at least 30 min to ensure complete reaction then activated
charcoal (Darco G—60, 720 g) and Celite (720 g) are added and stirred for at least 2 h. The
mixture is filtered washing the solid pad with EtOAc (2 x 1.8 L). The filtrate is concentrated
to afford tert-butyl N—tert-butoxycarbonyl—N—[5—br0m0—3-((trimethylsilyl)ethynyl) pyrazin
yl]carbamate (Compound C-3) that is used directly in the next step.
PCT/U32012/058127
Ste 3: Pre n of u 'lN— 5—bromoethvnvi vrazin-Z-vl
butoxycarbonylcarbamate {Compound Aii)
K2CO3 (811 g, 5.87 mol) is charged to a reactor followed by a solution of
Compound O3 (2300 g, 4.89 mol) dissolved in EtOAc (4.6 L) agitation started. EtOH (9.2 L)
is added slowly and the mixture stirred for at least 1 h to ensure that the reaction is complete
then water (4.6 L) is added and d for at least 2 h. The solid is ted by filtration and
washed with 1:1 EtOH/water (4.6 L followed by 2.3 L) followed by EtOH (2.3 L). The filter
cake is dried to afford zen—butyl N—(S—bromo~3—ethynylpyrazinyl)~N—tert—
butoxycarbonylcarbamate und A-4—ii) (1568 g, 78% yield, 97.5 area % by HPLC).1H
NMR (400 MHz, CDC13) 5 8.54 (s, 1H), 3.52 (s, 1H), 1.42 (s, 1811).
Solid Forms of Compound I—2
Compound I-2 has been prepared in various solid forms, including salts, and co-
solvates. The solid forms of the t invention are useful in the manufacture of
ments for the treatment of . One embodiment provides use of a solid form
described herein for treating cancer. In some embodiments, the cancer is pancreatic cancer or
non—small cell lung cancer. Another embodiment provides a pharmaceutical composition
comprising a solid form described herein and a pharmaceutically acceptable carrier.
Applicants describe herein five novel solid forms of Compound 1—2. The names
and stoichiometry for each of these solid forms are provided in Table 8—1 below:
- Table 3-1
Exam-1e
Examle l3 Comound 1—2 free base
Examle 14 Comoound I—2 - HCl
Example 15 Compound I-2 - 2HC1 12.
Examle 16 Comoound I-2 ° HCl ' H20 1:2:1
Examle l7 Comound I-2 ° HCl - 2H20 1:1:2
Solid state NMR spectra were acquired on the Bruker-Biospin 400 MHz
Advance III wide—bore spectrometer equipped with Bruker~Biospin 4mm HFX probe.
Samples were packed into 4mm ZrOz rotors ximately 70mg or less, depending on
sample availability). Magic angle spinning (MAS) speed of typically 12.5 kHz was applied.
The ature of the probe head was set to 275K to minimize the effect of onal
heating during spinning. The proton relaxation time was measured using lH MAS T1
saturation recovery relaxation experiment in order to set up proper recycle delay of the C
13C CPMAS experiment
cross—polarization (CP) MAS experiment. The recycle delay of was
WO 9726 PCT/U$2012/058127
adjusted to be at least 1.2 times longer than the measured lH T1 relaxation time in order to
maximize the carbon spectrum signal-to-noise ratio. The CP contact time of 13C CPMAS
experiment was set to 2 ms. A CP proton pulse with linear ramp (from 50% to 100%) was
employed. The Hartmann-Hahn match was optimized on external reference sample
(glycine). Carbon spectra were acquired with SPINAL 64 decoupling with the field strength
of approximately 100 kHz. The chemical shift was referenced t external standard of
adamantane with its upfield resonance set to 29.5 ppm.
XRPD data for Examples 13—14 were measured on Bruker D8 Advance System
(Asset V014333) equipped with a seal ed tube Cu source and a Vantec—l detector (Bruker
AXS, Madison, WI) at room temperature. The X—ray generator was operating at a voltage of
40 kV and a current of 40 mA. The powder sample was placed in a w silicon holder.
The data were ed in a reflection scanning mode (locked coupled) over the range of 3°—
40° 2 theta with a step size of 00144" and a dwell time of 0.253 (105 s per step). Variable
divergence slits were used.
Example 10: Compound I-2 (free base)
Compound 1-2 free base can be formed according to the methods described in Example 6,
Step 4: ate Method 1.
XRPD of Compound I—2 (free base)
Figure la shows the X—ray powder diffractogram of the sample which is
characteristic of crystalline drug substance.
] Representative XRPD peaks from Compound I-2 free base:
XRPD Angle Intensity %
Peaks (2-Theta :1: 0.2)
~ 23.8 100.0
*2 14.2 43.9
3 22.5 . 39.3
19.3 28.6
6 27.2 27.6
7 17.0 25.4
18.1 25.2
9 17.6 19.6
20.2 17.2
12 20.8 14.5'
13 29.9 14.5
33.2 14.3
.1 13.5
2012/058127
XRPD Angle Intensity %
Peaks (2-Theta :1: 0.2)
.6
.6
Thermo Analysis of Compound 1-2 free base
A thermal gravimetric analysis of Compound 1—2 free base was performed to
determine the percent weight loss as a on of time. The sample was heated from
t temperature to 350°C at the rate of 10°C/min on TA Instrument TGA Q5000 (Asset
V01425 8). Figure 2a shows the TGA result with a one-step weight loss before evaporation or
thermal decomposition. From ambient temperature to 215°C, the weight was ~l .9 %.
Differential Scanning Calorimetry of Compound I-2 free base
The thermal properties of Compound 1-2 free base were measured using the TA
Instrument DSC Q2000 (Asset 9). A Compound I—2 free base sample (1.6900 mg)
was weighed in a pre—punched pinhole aluminum hermetic pan and heated from ambient
temperature to 350°C at 10°C/min. One endothermic peak is observed at 210°C with its
onset ature at 201°C (Figure 3a). The enthalpy associated with the endothermic peak
is 78 J/g.
Solid State NMR of Compound I—2 free base
13C CPMAS on Compound I—2 free base
275K; 1H Tl=l.30s
12.5 kHz spinning; ref. adamantane 29.5 ppm
For the full spectrum, see Figure 4a.
W0 20131049726 2012/058127
Representative Peaks
um rel
l_——
126.6 100.0
' 123.5
38.1
Crystal Structure of nd 1-2 free base
The free form of Compound 1-2 was prepared from the Compound 1—2 HCl salt.
200mg Compound 1—2 HCl salt was added to lmL of 6N NaOH solution. 20mL of
dichloromethane was used to extract the free form. The dichloromethane layer was dried
over K2C03. The solution was filtered off and SmL of ane was added to it. Crystals
were obtained by slow evaporation of the solution at room temperature over night.
Most crystals obtained were thin plates. Some prismatic shape crystals were
found among them.
A yellow tic crystal with dimensions of 0.2>< 0.1><0.l mm3 was selected,
mounted on a MicroMount and centered on a Bruker APEX II diffractometer. Three batches
of 40 frames separated in reciprocal space were obtained to e an orientation matrix and
initial cell parameters. Final cell parameters were obtained and refined after data collection
was completed based on the full data set.
A diffraction data set of reciprocal space was obtained to a resolution of
11696" 20 angle using 0.5" steps with 10 s exposure for each frame. Data were collected at
100 (2) K temperature with a nitrogen flow cryosystem. Integration of intensities and
refinement of cell ters were accomplished using APEXII software.
PCT/U52012/058127
CRYSTAL DATA
Example 11: Compound I-2 - HCl
Compound 1-2 ° HCl can be formed according to the methods described in
Example 6, Step 4: Alternate Method 2 and Example 6, Step 5.
XRPD ofCompound I-2 - HCl
Figure 1b shows the X—ray powder diffractogram of the sample which is
characteristic of lline drug substance.
Representative XRPD peaks from Compound I—2 ' HCl
XRPD Angle ity %
Peaks (2—Theta :l: 0.2)
16.4
23.6
.5
.4
Thermo Analysis of Compound I—2 - HCl
A thermal gravimetric analysis of Compound 1—2 - HCl was performed to
determine the percent weight loss as a function of time. The sample was heated from
ambient ature to 350°C at the rate of 10°C/min on TA Instrument TGA Q5000 (Asset
W0 2013f049726 2012/058127
V01425 8). Figure 2b shows the TGA result with a two—step weight loss before evaporation
or thermal decomposition. From ambient temperature to 100°C, the weight was ~l .1 %, and
from 110°C to 240°C the weight loss is ~0.8%.
Differential Scanning Calorimetry of Compound 1—2 0 HCl
The thermal properties of Compound I~2 ° HCl were measured using the TA
Instrument DSC Q2000 (Asset V014259). A Compound I—2 - HCI sample (3.8110 mg) was
weighed in a pre—punched e aluminum hermetic pan and heated from ambient
temperature to 350°C at 10°C/min. One endothermic peak is observed at 293°C with its
onset temperature at 291°C (Figure 3b). The enthalpy associated with the endothermic peak
is 160.3 J/g. The second endothermic peak is around 321°C. Both peaks were d with
sample ation and decomposition.
Solid State NMR of Com ound 1-2 - HCl
CPMAS
on Compound 12 - HCl
275K;_12.5 kHz spinning; ref. adamantane 29.5 ppm
For the full spectrum, Sfi Figure 4b.
Representative Peaks
Peak Chem Shift Intensity
# o um rel
142.9 54 14
138.7 44 06
136.7 60 O6
129.8 73.58
127.9 63.71
124.1 34.91
53.52
W0 2013f049726 PCT/U82012/058127
Crystal Structure of Compound l-2 - HCl
] 180mg nd l-2 ° HCl was added to a vial with 0.8mL 2-propanol and 0.2
mL water. The sealed vial was kept in an oven at 70°C for two weeks. Diffraction quality
crystals were observed.
A yellow needle shape c1ystal with dimensions of 0.15X 0.02X0.02 mm3 was
selected, mounted on a MicroMount and centered on a Bruker APEX II ctometer
(V011510). Three s of 40 frames separated in reciprocal space were obtained to
provide an ation matrix and initial cell parameters. Final cell parameters were collected
and refined was completed based on the full data set.
A diffraction data set of reciprocal space was obtained to a resolution of 106° 29
angle using 05" steps with exposure times 20 s each frame for low angle frames and 603 each
frame for high angle frames. Data were ted at room temperature.
To obtain the data in table 1, dry nitrogen was blown to the crystal at 6
Litre/min speed to keep the ambient moisture out. Data in table 2 was obtained without
nitrogen. Integration of intensities and refinement of cell parameters were conducted using
the APEXII software. The water occupancy can vary between 0 and l.
masosoaa
V= 2510.87 (17) A3 v = 2527.2 (4) A3
CHN Elemental Analysis
CHN elemental analysis of Compound 1—2 - HCI suggest a mono HCl salt.
mesons
%Theory
57.60 5.20 14.00 7.10
%Foooo so-sa sass
Example 12: Compound I-2 - 2HC1
Compound I-2 ° ZHCI can be formed according to the s described in
Example 6, Step 4.
XRPD ofCompound I—2 - ZHCl
The XRPD patterns are acquired at room ature in reflection mode using a
Bruker D8 Discover system (Asset Tag V012842) equipped with a sealed tube source and a
Hi—Star area detector (Bruker AXS, Madison, WI). The X-Ray generator is operated at a
voltage of40 kV and a current of 35 mA. The powder sample is placed in a nickel holder.
Two frames are registered with an exposure time of 120 3 each. The data is subsequently
integrated over the range of 4.5°—39° 2 theta with a step size of 002° and merged into one
continuous pattern.
Figure 1c shows the X—ray powder diffractogram of the sample which is
characteristic of crystalline drug substance.
Representative XRPD peaks from Compound I—2 ° 2HC1
Peaks a :t 0.2)
100.0
92.2
91.3
91.3
91.2
_—89.0
89.0
l=-_88.8
9 88.1
*10 87.5
11 87.4
12 86.6
*13 86.0
14 86.0
86.0
85.9
85.9
85.7
85.7
85.4
85.2
85.2
84.9
84.7
84.1
PCT/U82012/058127
Thermo Analysis of Compound 1—2 ' 2HC1
A thermal etric analysis of Compound I-2 ' 2HC1 was performed on the
TA Instruments TGA model Q5000. nd I-2 ° 2HCl was placed in a um sample
pan and heated at 10°C/min to 350°C from room temperature. Figure 20 shows the TGA
result, which demonstrates a weight loss of 7.0% from room temperature to 188°C, which is
consistent with the loss of 1 equivalent of HCl (6.8%). The onset ature of
degradation/melting is 263°C.
Differential Scanning Calon’metg}: of Compound l~2 ° 2HC1
A DSC thermogram for Compound 1—2 - 2HC1 drug substance lot 3 was
obtained using TA Instruments DSC Q2000. Compound 1-2 - ZHCI was heated at 2°C/min to
275°C from —20°C, and modulated at i 1°C every 60 sec. The DSC thermogram (Figure 3c)
reveals an endothermic peak below 200°C, which could corresponds to the loss of 1
equivalent of HCl. Melting/recrystallization occurs between 215—245°C, followed by
degradation.
Solid State NMR of Compound I-2 - 2HC1
13c CPMAS on Compound 1-2 - 2HCl
275K; 1H T1=l.7s
12.5 kHz spinning; ref. adamantane 29.5 ppm
For the full spectrum, s_e§ Figure 4c.
Chem Shift um
Cmstal Structure of Compound I—2 ' 2HCl
180mg Compound I—2 ' HCl was added to a vial with 0.8mL anol and 0.2
mL water. The sealed Vial was kept in an oven at 70°C for two weeks. Diffraction quality
crystals were observed.
A yellow needle shape crystal with dimensions of 0. 15X 0.02 X0.02 mm3 was
selected, mounted on a MicroMount and ed on a Bruker APEX II diffractometer
). Three batches of 40 frames separated in reciprocal space were obtained to
provide an orientation matrix and initial cell parameters Final cell parameters were collected
and refined was completed based on the full data set.
A diffraction data set of reciprocal space was obtained to a tion of 106° 20
angle using 0.5° steps with exposure times 20 s each frame for low angle frames and 605 each
frame for high angle frames. Data were collected at room temperature. Dry nitrogen was
blown to the crystal at 6 Litre/min speed to keep the ambient moisture out. Integration of
intensities and ent of cell parameters were conducted using the APEXH software.
CRYSTAL DATA
V= 2510.87 (17) A3
Example 13: Compound I-2 - HCl 0 H20
Compound I—2 - HCl- H20 can be formed from Compound 1—2 - 2 HCl.
(E29244—17) A suspension of Compound I—2 - 2 HCl (10.0 g, 18.6 mmol) in isopropyl
alcohol (40 mL) and water (10 mL) is warmed at 50 °C for about 1 h and then cooled to
below 10 °C. The solid is ted by ion. The filter-cake is washed with 80/20
isopropyl alcohol/water (2X 10 mL) and air—dried to afford Compound I-2 - HCl- 2H20 as a
yellow powder.
PCT/U82012/058127
XRPD of Compound I-2 - HCl - HZPQ
The XRPD patterns are acquired at room temperature in reflection mode using a
Bruker D8 Discover system (Asset Tag V012842) equipped with a sealed tube source and a
Hi—Star area detector (Bruker AXS, Madison, WI). The X-Ray generator is operated at a
e of40 kV and a current of 35 mA. The powder sample is placed in a nickel holder.
Two frames are registered with an exposure time of 120 5 each. The data is subsequently
integrated over the range of 4.5°—39° 2 theta with a step size of 0.02° and merged into one
continuous pattern.
] Figure 1d shows the X—ray powder diffractogram of the sample which is
characteristic of lline drug nce.
Representative XRPD peaks from Compound l-2 - HCl - H20
XRPD Angle Intensity %
Peaks (2—Theta :l: 0.2)
*1—n_
17-7
11 31-1
12 15-8
14 17-1
12.7
16 16-0
18 20.6 16.0
*20 11.2 15.2
21 33.9 11.3
Thermo Analysis of Compound l—2 - HCl ° H20
Thermogravimetric analysis (TGA) for Compound 1-2 - l-lCl - H20 was
performed on the TA Instruments TGA model Q5000. Compound I—2 - HCl 0 H20 was
placed in a platinum sample pan and heated at 10°C/min to 400°C from room temperature.
The gram (Figure 2d) demonstrates a weight loss of 2.9% from room temperature to
PCT/U52012/058127
100°C, and a weight loss of 0.6% from 100°C to 222°C, which is consistent with theoretical
monohydrate (3.5%).
Differential Scanning Calorimetry of Compound 1-2 - HCl - H29
A DSC thermogram for Compound 1-2 ° HCI - H20 was obtained using TA
Instruments DSC Q2000. nd 12 - HCl ° H20 was heated at 2°C/min to 275°C from —
°C, and modulated at :r 1°C every 60 sec. The DSC gram (Figure 3d) reveals an
endothermic peak below 200°C, which could corresponds to the loss of 1 equivalent of HCl.
Melting/recrystallization occurs between 215-245°C, followed by degradation.
Example 14: Compound I-2 - HCI - 2H20
Compound 1—2 - HCl- 213120 can be formed from Compound I-2 ° 2 I-ICl.
(E29244—17) A suspension of Compound 1—2 - 2 HCl (10.0 g, 18.6 mmol) in isopropyl
alcohol (40 mL) and water (10 mL) is warmed at 50 °C for about 1 h and then cooled to
below 10 °C. The solid is collected by filtration. The filter-cake is washed with 80/20
pyl alcohol/water (2 x 10 mL) and ied to afford Compound 1—2 - HC1° 2H20 as a
yellow powder.
XRPD of Compound 1-2 - 1-1C1 0 21-129
The powder x—ray diffraction measurements were performed using
PANalytical’s X—pert Pro diffractometer at room temperature with copper radiation (1.54060
°A). The incident beam optic was comprised of a variable divergence slit to ensure a constant
illuminated length on the sample and on the diffracted beam side, a fast linear solid state
detector was used with an active length of 2. 12 degrees 2 theta measured in a ng mode.
The powder sample was packed on the indented area of a zero background silicon holder and
ng was performed to e better statistics. A symmetrical scan was measured from 4
~ 40 degrees 2 theta with a step size of 0.017 degrees and a scan step time of 15.53.
] Figure 1d shows the X—ray powder diffractogram of the sample which is
characteristic of crystalline drug substance.
] Representative XRPD peaks from Compound 1—2 - HCl - 2H20
XRPD Angle (2- Intensity %
Peaks Theta i 0.2)
100.0
82.0
69.2
66.3
63.8
‘i_ 5:33
.3- 51.8
9 20.7 49.7
15.3 46.2
44.6
41.2
19.9 40.4
14 17.6 39.1
39.0
36.1
33.6
33.5
32.2
29.1
28.7
28.0
23.8
22.6
26 14.3 22.4
.8
28 25.1 19.7
29 I 13.7 19.0
UJUJUJUJWUJUJUJUJUJ \OOOQOM-PUJN—‘O 14.0 17.4
33.0 16.2
9(- 23.3 15.7
16.6 15.1
29.6 14.9
29.9 14.8
27.6 14.8
32.1 13.3
-X- 24.6 13.1
.8 11.1
1 O4
W0 2013l049726 PCT/U82012/058127
Thermo Analysis of Compound 1-2 - HC1° 2HZQ
] The TGA (Thermogravimetric Analysis) thermographs were obtained using a
TA ment TGA Q500 respectively at a scan rate of lO°C/min over a ature range
of 25—300°C. For TGA analysis, samples were placed in an open pan. The thermogram
demonstrates a weight loss of~6 from room temperature to 100°C, which is consistent with
theoretical dihydrate (6.7%).
Differential Scanning Calorimetm of Compound I—2 - HCl ' 2H__20
A DSC (Differential ng Calorimetry) graphs were obtained using
a TA instruments DSC Q2000 at a scan rate of 10°C/min over a temperature range of 25~
300°C. For DSC analysis, samples were weighed into aluminum hermetic T-zero pans that
were sealed and punctured with a single hole. The DSC thermogram reveals ation
between room temperature and 120°C followed by g/recrystallization between 170—
250°C.
Crystal Structure of Compound 1—2 - HCl with water
180mg Compound I-2 - HCl was added to a vial with 0.8mL 2—propanol and 0.2 mL water.
The sealed vial was kept in an oven at 70°C for two weeks. Diffraction quality crystals were
A yellow needle shape crystal with dimensions of 0.15>< 0.02><0.02 mm3 was
ed, mounted on a MicroMount and centered on a Bruker APEX II diffractometer
(V011510). Then a kapton tube with water inside covered the pin. The tube was sealed to
make sure the crystal is equilibrated with water for two days before the diffraction
experiments. Three batches of 40 frames separated in reciprocal space were obtained to
provide an orientation matrix and initial cell parameters Final cell ters were collected
and refined was completed based on the full data set.
A diffraction data set of reciprocal space was obtained to a resolution of 106° 20
angle using 0.5° steps with exposure times 20 3 each frame for low angle frames and 60s each
frame for high angle frames. Data were collected at room temperature. Integration of
intensities and refinement of cell parameters were conducted using the APEXII software.
PCT/U52012/058127
CRYSTAL DATA
Example 15: Cellular ATR Inhibition Assay:
Compounds can be screened for their ability to inhibit intracellular ATR using
an immunofluorescence copy assay to detect phosphorylation of the ATR substrate
histone H2AX in hydroxyurea treated cells. HT29 cells are plated at 14,000 cells per well in
96—well black imaging plates (BD ) in McCoy’s 5A media (Sigma M8403)
supplemented with 10% foetal bovine serum (JRl-I ences 12003),
Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2mM L—glumtamine
(Sigma G7513), and allowed to adhere overnight at 37°C in 5% C02. Compounds are then‘
added to the cell media from a final concentration of 25uM in 3-fold serial dilutions and the
cells are incubated at 37°C in 5% C02. After 15min, hydroxyurea (Sigma H8627) is added to
a final concentration of 2mM.
After 45min of treatment with hydroxyurea, the cells are washed in PBS, fixed
for 10min in 4% formaldehyde diluted in PBS (Polysciences Inc 18814), washed in 0.2%
Tween—20 in PBS (wash buffer), and permeabilised for 10min in 0.5% Triton X-100 in PBS,
all at room temperature. The cells are then washed once in wash buffer and blocked for
30min at room temperature in 10% goat serum (Sigma G9023) diluted in wash buffer (block
buffer). To detect H2AX phosphorylation levels, the cells are then incubated for 1h at room
ature in primary antibody (mouse onal hosphorylated histone H2AX
Serl39 antibody; Upstate 05-636) diluted 1:250 in block . The cells are then washed
five times in wash buffer before incubation for 1h at room temperature in the dark in a
mixture of secondary antibody (goat anti-mouse Alexa Fluor 488 conjugated antibody;
Invitrogen A11029) and Hoechst stain (lnvitrogen H3570); diluted 1:500 and 1:5000,
respectively, in wash buffer. The cells are then washed five times in wash buffer and finally
100ul PBS is added to each well before imaging.
2012/058127
Cells are imaged for Alexa Fluor 488 and Hoechst intensity using the BD
Pathway 855 Bioimager and Attovision software (BD Biosciences, Version 1.6/855) to
quantify phosphorylated H2AX Serl39 and DNA staining, respectively. The percentage of
phosphorylated H2AX-positive nuclei in a montage of 9 images at 20x magnification is then
calculated for each well using BD Image Data er software (BD Biosciences Version
2.2.15). Phosphorylated H2AX-positive nuclei are defined as Hoechst—positive regions of
interest containing Alexa Fluor 488 intensity at 1.75—fold the e Alexa Fluor 488
intensity in cells not treated with hydroxyurea. The percentage ofH2AX positive nuclei is
finally plotted against concentration for each nd and ICSOs for intracellular ATR
inhibition are determined using Prism re (GraphPad Prism n 3.ch for
Macintosh, GraphPad Software, San Diego California, USA).
The nds bed herein can also be tested according to other methods
known in the art (5% Sarkaria et a1, “Inhibition ofATM and ATR Kinase Activities by the
Radiosensitizing Agent, Caffeine: Cancer Research 59: 4375—5382 (1999); Hickson et al,
“Identification and Characterization of a Novel and c Inhibitor of the Ataxia-
Telangiectasia Mutated Kinase ATM” Cancer Research 64: 159 (2004); Kim et a1,
“Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family
Members” The Journal ofBiological Chemistry, 274(53): 37538-37543 (1999); and Chiang
et a1, “Determination of the catalytic activities of mTOR and other members of the
phosphoinositide-3—kinase—related kinase family” Methods M01. Biol. 281 :125—41 (2004)).
e 16: ATR Inhibition Assay:
Compounds can be screened for their ability to inhibit ATR kinase using a
radioactive—phosphate incorporation assay. Assays are carried out in a mixture of SOmM
Cl (pH 7.5), lOmM MgClz and 1mM DTT. Final substrate concentrations are lOuM
[y—33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 uM target peptide
ASQPQPFSAKKK).
Assays are carried out at 25°C in the presence of 5 nM full—length ATR. An assay
stock buffer solution is prepared containing all of the reagents listed above, with the
exception of ATP and the test compound of interest. 13.5 pL of the stock solution is placed
in a 96 well plate followed by on of 2 pL of DMSO stock containing serial dilutions of
the test compound (typically starting from a final concentration of 15 uM with 3-fold serial
dilutions) in duplicate (final DMSO concentration 7%). The plate is pre-incubated for 10
minutes at 25°C and the reaction initiated by addition of 15 uL [y—33P]ATP (final
concentration 10 uM).
The reaction is stopped after 24 hours by the addition of 30uL 0.1M phosphoric
acid containing 2mM ATP. A multiscreen ocellulose filter 96-well plate (Millipore,
Cat no. MAPHNOBSO) is pretreated with IOOuL 0.2M phosphoric acid prior to the addition
of 45uL of the stopped assay mixture. The plate is washed with 5 x 200nL 0.2M phosphoric
acid. After drying, 100 uL ase Mix’ liquid scintillation cocktail (Perkin Elmer)
is added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation
Counter, Wallac).
After removing mean background values for all of the data , Ki(app) data are
calculated from non—linear regression analysis of the initial rate data using the Prism re
package (GraphPad Prism version 3.0cx for osh, GraphPad Software, San Diego
California, USA).
In general, the compounds of the present ion are effective for inhibiting ATR.
Compounds I-l, 1—2, 11—1, 11-2, 11-3 and 11—4 inhibit ATR at Ki values below 0.001 pM.
Example 17: Cisplatin Sensitization Assay
Compounds can be screened for their y to sensitize HCT116 colorectal cancer
cells to Cisplatin using a 96h cell ity (MTS) assay. HCTI 16 cells, which possess a
defect in ATM signaling to Cisplatin (s_e_e, Kim et al.; Oncogene 21 :3864 (2002); m,
Takemura et al.; JBC 281230814 (2006)) are plated at 470 cells per well in 96-well
polystyrene plates (Costar 3596) in 150g] of McCoy’s 5A media (Sigma M8403)
supplemented with 10% foetal bovine serum (JRH Biosciences 12003),
Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2mM L—glumtamine
(Sigma 67513), and allowed to adhere overnight at 37°C in 5% C02. nds and
Cisplatin are then both added simultaneously to the cell media in 2—fold serial dilutions from
a top final concentration of IOuM as a full matrix of concentrations in a final cell volume of
200ul, and the cells are then incubated at 37°C in 5% C02. After 96h, 40ul ofMTS t
(Promega G358a) is added to each well and the cells are incubated for 111 at 37°C in 5% C02.
Finally, absorbance is measured at 490nm using a SpectraMax Plus 384 reader (Molecular
Devices) and the concentration of compound required to reduce the ICSO of Cisplatin alone
by at least 3—fold (to 1 decimal place) can be reported.
WO 49726 2012/058127
Example 18: Single Agent HCT116 Activity
Compounds can be screened for single agent activity against HCT116 colorectal
cancer cells using a 96h cell viability (MTS) assay. HCTl 16 are plated at 470 cells per well
in 96—well polystyrene plates (Costar 3596) in 150m of McCoy’s 5A media (Sigma M8403)
supplemented with 10% foetal bovine serum (JRH Biosciences 12003), Penicillin/
Streptomycin solution diluted 1:100 (Sigma P7539), and 2mM L—glumtamine (Sigma
G7513), and allowed to adhere overnight at 37°C in 5% C02. Compounds are then added to
the cell media in 2—fold serial dilutions from a top final concentration of lOuM as a full
matrix of concentrations in a final cell volume of 2001,11, and the cells are then incubated at
37°C in 5% 002. After 96h, 40m ofMTS reagent (Promega G35 8a) is added to each well
and the cells are incubated for lh at 37°C in 5% C02. Finally, absorbance is ed at
490nm using a aMax Plus 384 reader (Molecular Devices) and IC50 values can be
calculated.
_________pl___esData for Exam 18-21
Cisplatin
sensitization
While we have described a number of embodiments of this invention, it is apparent
that our basic examples may be altered to provide other ments that utilize the
compounds, methods, and processes of this invention. Therefore, it will be appreciated that
the scope of this invention is to be defined by the ed claims rather than by the specific
embodiments that have been represented by way of example herein.
Claims (11)
1. A process for preparing Compound I-2•HCl: Compound I-2 comprising ng a suspension of Compound I-2•2HCl in i-PrOH and water.
2. A process for preparing Compound l: comprising combining about 1 equivalent of an aqueous solution of HCl with the free base of Compound I-2 in acetone.
3. A solid form of a compound of formula I-2: n the form is selected from Compound I-2•HCl, Compound I-2•HCl•H2O, Compound I-2•HCl•2H2O, and Compound I-2•2-hydrochloric acid.
4. The solid form of claim 3, wherein the form is Compound I-2•HCl.
5. The solid form of claim 4, wherein the form is crystalline Compound I-2•HCl.
6. The solid form of any one of claims 4-5, wherein the compound:HCl are in a ratio of 1:1.
7. The solid form of any one of claims 5-6, having a monoclinic crystal system, having a P21/n space group, and having the following unit cell dimensions in Å when measured at 120 K: a = 5.3332 (2) Å b = 35.4901 (14) Å c = 13.5057 (5) Å.
8. The solid form of any one of claims 5-7, characterized by a weight loss of from about 1.1 % in a temperature range of from about 25 °C to about 100 °C, and about 0.8 % from 110 °C to about 240 °C.
9. The solid form of any one of claims 5-8, characterized by one or more peaks expressed in 2-theta ± 0.2 at about 13.5, 28.8, 15.0, 18.8, and 15.4 degrees in an X-ray powder ction pattern obtained using Cu K alpha radiation.
10. The solid form of any one of claims 5-9, characterized by one or more peaks at about 171.7, 153.4, 132.9, 31.8, and 15.7 ppm in solid state 13 CNMR.
11. The solid form of any one of claims 5-10, characterized as having an X-ray powder diffraction pattern substantially the same as that shown in
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