Process Development and Large-Scale Synthesis of BTK Inhibitor BIIB068

Article abstract of DOI:10.1021/acs.oprd.0c00087

Chemical process development efforts leading to multikilogram production of BIIB068 hemiadipate are discussed. Process optimization resulted in (1) removal of transition metal from the process, (2) a streamlined process with significantly improved overall

Full text of DOI:10.1021/acs.oprd.0c00087

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Communication
Process Development and Large-Scale Synthesis of BTK Inhibitor BIIB068
Chaomin Li, Lloyd Franklin, Robbie Chen, Tamera Mack, Michael Humora, Bin Ma,
Brian T Hopkins, John Guzowski, Fengmei Zheng, Michael MacPhee, Yiqing Lin, Steven
Ferguson, Daniel Patience, George A. Moniz, William F Kiesman, and Erin M O'Brien
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00087 • Publication Date (Web): 21 Apr 2020
Downloaded from pubs.acs.org on April 21, 2020
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Process Development and Large-Scale Synthesis of BTK
Inhibitor BIIB068
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*
Chaomin Li, Lloyd Franklin, Robbie Chen, Tamera Mack, Michael Humora, Bin Ma, Brian T. Hopkins,
John Guzowski, Fengmei Zheng, Michael MacPhee, Yiqing Lin, Steven Ferguson, Daniel Patience,
George A. Moniz, William F. Kiesman, Erin M. O’Brien
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Abstract:
Chemical process development efforts leading to multi-kilogram production of BIIB068 hemi-
adipate will be discussed. Process optimization has resulted in 1) removal of transition metal
from the process; 2) a streamlined process with significantly improved overall yield; 3)
appropriate impurity control (including potential mutagenic impurities or PMI), which enabled
delivery of quality material for toxicology studies and clinical trials.
Keywords: Process development, BTK inhibitor, Amination, Urea formation, Impurity control,
Hemi-adipate
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Introduction
Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase that
functions downstream of the B cell receptor (BCR) in B cells and downstream of Fc receptors in
myeloid cells. BTK mediates B cell receptor signaling leading to regulation of B cell activation,
proliferation and differentiation. There is a strong biologic rationale for targeting diseases in
which the B cell pathway is central to disease pathogenesis such as B cell mediated autoimmune
diseases as well as B cell lymphoma or leukemia and atopic diseases. Ibrutinib is a clinically
1
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1
validated BTK inhibitor. In addition, inhibition of BTK kinase activity may also provide
therapeutic benefit to patients suffering from autoimmune disorders by blocking aberrant B and
myeloid cell activation.2
As part of an ongoing research/development program at Biogen, an
aminopyrazolopyrimidine compound BIIB068 (1), was discovered as a highly potent BTK
inhibitor with good oral bioavailability, and a promising candidate for the treatment of
autoimmune diseases with significantly unmet medical needs such as Systemic Lupus
Erythematosus (SLE) and primary Sjögren’s Syndrome (pSS).3
O
H
N
N
O
N
N
N
N
N
H
BIIB068 (1)
Figure 1. BIIB068 (1), a potent BTK inhibitor
While the discovery synthesis of BIIB068 was straightforward in terms of bond
construction strategy, there was significant room for reaction condition improvement to enable a
suitable process for large-scale production of drug substance to support preclinical and clinical
studies. This article describes process development efforts that resulted in a practical and
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scalable manufacturing process for multi-kilogram production of BIIB068 hemi-adipate co-
crystal (19).
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Results and Discussion
Medicinal Chemistry Synthetic Route to 1. Synthesis of 1 via the discovery route is shown in
Scheme 1. Borane reduction of 4-bromo-2-methylbenzonitrile (2) afforded benzylamine 3 which
was isolated as an HCl salt in 90 % yield. Boc protection of 3 gave 4 as a white solid which was
used in the next step without further purification with 95 % yield. Aryl bromide 4 was converted
to the corresponding boronate (5) via Miyaura borylation (69 % yield). Subsequent Suzuki
reaction of boronate 5 with 2,4-dichloropyrimidine (6) delivered biaryl intermediate 7 in 80%
yield after silica gel column purification. A transition metal catalyzed C-N coupling of 7 with 1-
methyl-1H-pyrazol-4-amine (8) was then applied to install the aminopyrazole moiety and the C-
N coupling product 9 was isolated in 63% yield using chromatography. The Boc protecting
group in 9 was successfully removed using HCl in MeOH to liberate the amino group and deliver
benzylamine 10 in 90 % yield. The last chemical transformation step in the 1 synthesis involved
urea formation with 3-isopropoxyazetidine (11) using CDI as acylating reagent to give 1 in 80-
8
5% yield after silica gel column chromatography.
Scheme 1. Synthesis of 1 via the discovery route
NHBoc
Cl
N
HCl
NH2
NHBoc
6
N
CN
a
b
c
Cl
B
d
O
O
Br
Br
Br
2
3
4
5
O
NHBoc
NH2
H
N
NHBoc
N
N
N
NH
O
O
H N
2
f
11
8
N
N
g
N
N
N
e
N
N
N
N
N
N
H
N
N
N
N
Cl
H
N
N
H
7
9
10
1
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Reagents and conditions: (a) BH , THF, 80 °C, 16 h, 90%; (b) Boc O, Et N, THF, 1 h, 95%; (c) B Pin ,
3
2
3
2
2
Pd(dppf)Cl , KOAc, 1,4-dioxane, 100 °C, 2 h, silica gel column chromatography, 69%; (d) 6,
2
Pd(dppf)Cl , K CO , 1,4-dioxane:H O (4:1), 90 °C, 2 h, silica gel column chromatography, 80%; (e) 8,
2
2
3
2
Pd (dba) , S-Phos, Cs CO , 1,4-dioxane, 120 °C, 2 h, silica gel column chromatography, 63%; (f) HCl,
2
3
2
3
MeOH, rt, 6 h, 90%; (g) 11, CDI, THF, rt, 48 h, silica gel column chromatography, 80-85%. Boc O = Di-
2
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tert-butyl dicarbonate; B Pin = Bis(pinacolato)diboron; Pd(dppf)Cl = [1,1′-
2
2
2
Bis(diphenylphosphino)ferrocene]dichloropalladium(II); Pd (dba) =
2
3
Tris(dibenzylideneacetone)dipalladium(0); S-Phos = 2-Dicyclohexylphosphino-2′,6′-dimethoxybiphenyl;
CDI = 1,1′-Carbonyldiimidazole
General Strategy for Process Development to Enable Large-Scale Synthesis. Although the
bond disconnection strategy of the medicinal chemistry route to 1 was largely a linear synthesis,4
it was determined that the discovery synthesis was suitable for the initial kilogram scale
preparation of 1 based on the following considerations: (1) timeline constraints to produce drug
substance and support toxicology studies, formulation development and Phase I clinical trials;
(2) large quantities (> 10 kg) of advanced intermediate 7 had been outsourced and were available
for GMP manufacturing; (3) the discovery synthesis was not thoroughly optimized leaving room
for process improvement. Therefore, our efforts were focused on the development of final three
steps (from 7 to 1). Our goals were to (1) improve reaction profiles and yields; (2) understand
and appropriately control the impurities generated in the process to meet the product quality
profile; (3) remove two silica gel column chromatography steps (step 7 to 9 and step 10 to 1) to
enable large-scale production.
Installation of aminopyrazole and in-situ Boc deprotection. The first step of the medicinal
chemistry route to 1 from intermediate 7 involved a C-N coupling with aminopyrazole 8 using a
Pd catalyst. As demonstrated by ample literature precedents, amination of 2-chloropyrimidines
5
6
can be accomplished under thermal conditions in the presence of either acids or bases, in
addition to transition metal catalyzed processes.7
1
) Reaction optimization: In the event, an initial screening study found that the reaction did not
8
9
require transition metal (Pd) catalysis or undesirable 1,4-dioxane as solvent (Table 1, entry 1).
Although the reaction condition using Cs CO without Pd catalyst afforded only 10% desired
2
3
product 9 (Table 1, entry 2), significant improvement (~ 50%) was realized when K CO was
2
3
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used as base in 1-butanol or NMP (Table 1, entries 4 and 5). When the base was changed to
organic bases such as DIEA or Et N, the reaction profiles were further improved to ~ 60 % of 9
3
with ~ 30% of 7 remaining (Table 1, entries 6 and 7).
Advantageously, it was found that under acidic conditions the reaction afforded the desired C-N
coupling product and had the added benefit of removing the Boc protecting group in a one-pot
process. Initial reaction conditions with 3 equiv of H PO in 1-butanol yielded 91% of the de-
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Boc product 10 (Table 1, entry 8). At this stage of optimization, we examined the HCl salt of 8
as an alternative coupling partner, anticipating the reaction would proceed without additional
acid. As expected, the reaction proceeded cleanly giving 92% of 10 (Table 1, entry 10). After
optimization of reaction conditions including solvent and temperature, we concluded that heating
8
.
HCl salt in 1-butanol at 85 °C was the optimal reaction condition and comparable with
reactions at 100 °C (Table 1, entry 11). In this case, the HCl from the 8 HCl salt and that
generated from the displacement reaction was sufficient to promote the Boc deprotection.
.
1
0
Formation of of butylated impurity (14, Figure 2) under various conditions (Table 1, entries 3
and 4) led us to switch from 1-butanol to 2-butanol. The butylated impurity was not detected
when the secondary alcohol was used as reaction solvent, although the reactivity in 2-butanol
was found to be lower compared with 1-butanol and 15% of 9 and 9% of 12 remained after 16 h
of reaction (Table 1, entry 12). After further optimization, the solvent mixture of 2-
butanol/water was found to afford better reactivity than 2-butanol alone (Table 1, entry 13), as
the binary solvent enabled a homogenous solution compared with heterogenous reaction mixture
when either 1-butanol or 2-butanol was used as single solvent. This homogenous binary solvent
system was also ideal for scale-up. The V/V ratio of 2-butanol / water was not critical for the
reaction performance and the reaction proceeded well using 2-butanol / water mixture with V/V
ratios ranging from 5:2 to 2:3. However, the amount of 2-butanol used had a significant impact
on the product loss to the mother liquor. Two volumes of 2-butanol (with respect to the weight
of 7) were used to maximize the isolated yield. An excess of 8
the reaction to completion. Initially 1.2 equivalents were used, but in an effort to accelerate the
reaction the amount of 8 HCl salt was increased to 1.3 equivalents. Under optimized reaction
conditions, different equivalents of 8 HCl salt were explored (Table 1, entries 13-15) and it was
.
HCl salt was necessary to drive
.
.
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concluded that 1.3 equivalents of 8 HCl salt were optimal as more did not give further
.
improvement of reaction profile.
Table 1: Screening results for the C-N coupling of 7 with 8a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
4
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
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NH2
NHBoc
NHBoc
NH2
N
N
H N
2
8
+
N
N
N
N
N
Conditions
N
N
N
N
N
H
N
Cl
N
N
H
N
Cl
7
12
9
10
Entry
1b
Reagents
Solvent
Temp 7 (HPLC A%) 9 (HPLC
10 (HPLC
A%)
A%)
Pd (dba) (0.2
1,4-dioxane 100 °C
2
58
0
2
3
equiv), S-
Phos(0.1 equiv),
Cs CO (2
2
3
equiv)
2
Cs CO (2
1,4-dioxane 100 °C
67
10
0
2
3
equiv)
3c
Cs CO (2
1-butanol
100 °C
< 1
< 1
< 1
2
3
equiv)
4d
5
K CO (2 equiv)
1-butanol
NMP
100 °C
100 °C
100 °C
100 °C
100 °C
100 °C
100 °C
85 °C
5
25
31
32
0
47
52
61
60
0
< 1
< 1
< 1
< 1
91
2
3
K CO (2 equiv)
2
3
6
DIEA (2 equiv)
1-butanol
1-butanol
1-butanol
1-butanol
1-butanol
1-butanol
2-butanol
7
Et N (2 equiv)
3
8
H PO (3 equiv)
3 4
9
AcOH (3 equiv)
None
0
43
0
54
1
0e
11e
2e,f
0
92
None
0
0
91
1
None
85 °C
< 1
15
71
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1
3e
None
None
None
2-
85 °C
85 °C
85 °C
0
0
0
0
0
0
95
94
93
butanol/water
(
2V/3V)
1
1
4e,g
2-
0
1
2
3
4
5
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8
9
0
1
2
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9
0
1
2
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8
9
0
1
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0
1
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9
0
butanol/water
(2V/3V)
5e,h
2-
butanol/water
(
2V/3V)
a1.3 equiv of 8 or 8
run for 16 h except for entries 1 and 2 for which the reactions aged for 2 h before analytical
HCl salt used for conditions except for entries 14 and 15. All reactions were
.
b
c
samples were taken. Crude reaction contained 15% of 12. Crude reaction contained 51% of 13
d
e
and 28% of 14. Crude reaction contained 39% of 14. 8
.
HCl salt instead of 8 free base was
f
g
used for the reactions. Crude reaction mixture contained 9 % of 12. 1.5 equiv of 8
.
HCl salt
h
used. 2.0 equiv of 8
.
HCl salt used.
NH2
N
N
NHBoc
NH2
NH
N
O
N
O
N
N
N
N
N
N
N
13
14
H
1
5
Table 1, entry 3
Table 1, enties 3 and 4
Figure 2. Impurities generated in amination reaction of 2-chloropyrimidine 7 with
aminopyrazole 8
2) Isolation and workup: During process development, it was observed that complete
consumption of 12 was necessary before performing the workup. Under basic workup
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conditions the chloride on unreacted 12 was displaced by the benzylic amine group of 10 to form
a dimer-like side product 15 that did not purge well in the subsequent step. After exploration of
various workup conditions, a simple precipitation of 10 free base through the addition of
ammonium hydroxide and water was discovered. The workup was originally performed at 20 °C
with the addition of ammonium hydroxide followed by dilution with water. This process was
complicated with free base 10 precipitating out quickly when the pH of the reaction mixture
changed from acidic to basic, resulting in a very thick mixture that was difficult to stir. In order
to alleviate this agitation problem, the temperature was increased to 70 °C for the workup, which
enabled mixing but resulted in the formation of an unidentified late-eluting impurity. The
optimal balance of workability and purity was achieved by decreasing the workup temperature to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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0
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0
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0
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0
1
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9
0
3
5 °C, diluting the reaction mixture with water and the free base was precipitated through
addition of ammonium hydroxide. Further optimization resulted in addition of 60% of the total
water amount to the reaction followed by addition of a mixture of ammonium hydroxide and the
remaining 40% of the water charge, which resulted in isolated 10 in 90 % yield and > 99 %
HPLC purity.
Urea formation leading to synthesis of 1. After optimizing the synthesis of benzylamine 10,
we turned our attention to the final chemical bond construction step: the urea formation to
generate 1 (Scheme 2).
Scheme 2. Urea synthesis to generate 1
O
NH2
H
N
N
NH
O
O
11
N
N
N
CDI
N
N
N
N
N
H
N
N
H
1
10
1) Azetidine: During development, selection of appropriate form of azetidine starting material
was deemed important. 3-Isopropoxyazetidine (11) is available as either a solid HCl salt or a
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liquid free base. Both forms can be used in the current process; however, the free base provided
several advantages over the HCl salt. First, the reaction was noticeably faster with the free base.
Second, the HCl salt is extremely hygroscopic and requires special handling. Third, the HCl salt
is charged to the reaction as a DMSO solution, while the liquid free base can be conveniently
charged into the reaction directly. Finally, a higher level of the chloride ring-opening impurity 16
(Figure 3) was observed when using HCl salt for the reaction suggesting the reaction of 1 with
HCl in the matrix. For the current process the use of 3-isopropoxyazetidine (11) free base was
therefore chosen for development.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
O
H
H
H
N
H
N
N
N
N
NH
Cl
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
N
N
H
H
H
18
16
17
Figure 3: Impurities generated during urea formation step
) Reaction solvent: The urea formation reaction proceeded in a variety of solvents. Polar
2
aprotic solvents were chosen for scale-up as they fully dissolved all materials and intermediates
and eliminated potential stalling observed in some heterogeneous reactions. Although THF was
used in the medicinal chemistry route, early toxicology lots were synthesized using two solvents:
N,N-dimethylformamide (DMF) and dimethylacetamide (DMAc). An impurity associated with
DMF (17, Figure 3) was tentatively identified by LC-MS; therefore, the solvent was changed to
DMAc. However, use of DMAc as the reaction solvent promoted the formation of a bis-addition
product 18 (Figure 3). After production of the early toxicology lots, dimethylsulfoxide (DMSO)
was determined to be a more suitable solvent providing a cleaner reaction (Table 2, entry 5) with
an ICH guideline Class 3 solvent. All 1 prepared for the IND-enabling toxicology studies and
clinical studies used DMSO as the solvent for the urea formation step.
3
) Bis-addition impurity 18: Unlike impurities 16 and 17, which can be avoided by using
alternative reactants or solvents, a small amount (as low as <1 %) of impurity 18 was observed in
all reactions. The impurity level of 18 was linked to 1) the order of addition of 10 and CDI and
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) the CDI equivalents. When a solution of CDI was added to a solution of 10, impurity 18 was
formed in larger amount (Table 2, entries 1-4). The impurity level of 18 was reduced by adding
the solution of 10 to the CDI solution over one hour at 30-40°C. Compound 1 made for the
IND-enabling toxicology studies and clinical studies used this procedure.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2: Optimization of urea formation step to minimize impurity 17 and 18a
Entry Solvent Reaction conditions for Product 1b
Impurity 17 Impurity 18
mixing 10 with CDI
1.2 equiv of CDI in
DMF solution was
added to 10 in DMF
10 in DMF solution was
added to 1.2 equiv of
CDI in DMF
(HPLC A%) (HPLC A%) (HPLC A%)
1
2
3
4
5
6
DMF
DMF
92.7
94.3
73.6
86.5
95.8
96.5
1.8
1.9
0
1.3
0.7
21
DMAc
DMAc
DMSO
DMSO
0.8 equiv of CDI in
DMAc solution was
added to 10 in DMAc
10 in DMAc solution
0
10.2
1.4
0.8
was added to
0.8
equiv of CDI in DMAc
10 in DMSO solution
0
was added to
0.9
equiv of CDI in DMSO
10 in DMSO solution
0
was added to
1
equiv of CDI in DMSO
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DMSO
10 in DMSO solution
96.6
0
0.6
was added to 1.2
equiv of CDI in DMSO
a: Reactions were all carried out at 30 °C first by mixing 10 and CDI for < 1 h and then followed by
charging 11 and aging for 5 h. b: < 0.5 % HPLC A% of 10 was found in all of the crude reaction
mixtures.
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Impurity 18 was also formed in much greater quantities when CDI was undercharged.
When 0.8 equivalent of CDI was used, the impurity level of 18 reached as high as ~20 % (Table
2, entry 3). Generation of impurity 18 was also observed if CDI decomposed slightly before use.
To mitigate this, CDI exposure to air or water was avoided and CDI was stored under
refrigeration and an inert atmosphere of nitrogen. On manufacturing scale, the best packaging
found to maintain reagent integrity was in unopened, heat sealed, UV protected bags stored
under refrigeration. To ensure quality, an extra bag of the reagent having the same lot number
was purchased for use in a pre-production use test. The amount of CDI was optimized with
DMSO as solvent, it was found that 1.2 equivalent of CDI was sufficient to drive the reaction to
completion without significant amount of impurity 18 formation (Table 2, entries 5-7).
Impurity 18 can be removed from 1 by dissolving crude 1 in 10 volumes of ethanol at
reflux. The insoluble impurity is removed by hot filtration. This procedure was incorporated in
the final co-crystal formation step (see below).
The optimized urea formation procedure was demonstrated at > 100 g scales generating 1
in > 95 % isolated yield and > 99 % HPLC purity.
1 Hemi-Adipate Co-Crystal. As the output of pharmaceutical property assessments with the
main goal of improving bioavailability, two salt forms of 1 were identified and characterized as
potentially suitable solid forms for further development: the HCl salt and the hemi-adipate co-
1
1
crystal (19). Initial development efforts focused on the HCl salt, however, the chloro-impurity
1
2
16 was generated when 1 was treated with HCl. DEREK analysis suggested compound 16 is a
potential mutagenic impurity (PMI) and efforts were therefore shifted to the development of the
hemi-adipate co-crystal 19. 13
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Scheme 3: 1 Hemi-Adipate co-crystal formation
O
O
H
N
H
N
N
N
O
O
O
HO
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
adipic acid
OH
0.5
O
N
N
N
N
N
N
N
H
N
N
H
BIIB068 Hemi-Adipate Co-Crystal (19)
BIIB068 Free Base (
1)
Both the hemi-adipate co-crystal and mono-adipate co-crystal can be produced. The co-
crystals appear loosely bound and both require an excess of adipic acid to crystallize. It was
found that isolation of the hemi co-crystal was favored when 0.9-1.1 equiv of adipic acid was
used, while the mono-adipate co-crystal was favored with 1.9-2.1 equiv of adipic acid. The
hemi-adipate co-crystal was selected for further development.
In the early process evaluation phase, the hemi-adipate co-crystal was readily prepared
and precipitated directly via primary nucleation. However, during the development process the
purity profile and the composition of the feed stream to the crystallization/co-crystal formation
step changed. The new impurity profile inhibited primary nucleation of hemi-adipate, which then
could only be produced via seeding and secondary nucleation. To ensure a successful campaign,
seeded co-crystal formation was therefore used for production.
The hemi-adipate co-crystal was initially produced in either THF and ethanol. Yield,
purity and processing were similar for both solvents. To develop a practical co-crystal formation
process, solubility screening was conducted and both 1 and its hemi-adipate (19) exhibited high
solubility in polar non-protic solvents such as DMSO, DMF and NMP, however, these solvents
were found to facilitate disproportionation. Moderate solubility (highly temperature-dependent)
was observed in alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol), 1,4-dioxane,
THF, dichloromethane and methyl ethyl ketone. Overall, the solubility screen for 1 and 19
showed alcohol systems (e.g.1-propanol, ethanol, 1-propanol/ethanol mixture) to be most
promising for further crystallization development of 1 hemi-adipate. The solubility of both 1 and
1
9 was acceptable for scale-up, with 19 showing ca. 90-120 mg/mL at 70 °C and less than 10
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mg/mL at 5 °C, making a cooling crystallization process feasible. Therefore, ethanol, a Class 3
solvent, was selected for further development.
When ethanol was used as solvent, an ethyl carbamate impurity 20 was generated in the
hemi-adipate process. Control experiments found that the unreacted urea intermediate 21 (from
urea formation step) in 1 reacts with ethanol in the co-crystal formation step to generate
carbamate 20. To minimize formation of impurity 20 from 21, the urea formation step was
modified to include a residual limit of <0.1% of 21 as well as an increased reaction time or an
additional azetidine charge to achieve this limit. Additional lab experiments suggested that 1
also reacted with ethanol to form impurity 20 at a rate of ~ 0.002% per hour at 75 °C. The
reaction rate increased significantly with the addition of adipic acid. When adipic acid was
present, impurity 21 formed at a faster rate of ~0.01% per hour at 75 °C. To minimize
formation of impurity 20 from 1, the GMP co-crystal formation process was modified so that
instead of combining the materials and heating, 1 and the adipic acid were heated separately in
ethanol to 75 °C and then combined.
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4: Formation pathway of ethyl carbamate impurity 20
N
O
H
N
H
N
H
N
O
N
N
O
O
O
EtOH
EtOH
N
N
N
N
N
N
N
N
N
N
N
H
N
N
H
N
N
H
21
20
1
A consistent and reproducible process was thus developed. The reaction was carried out
at >70 °C, followed by seeding and crystallization at 60 °C and ramp cooling to 0 °C. The
product was isolated by filtration. Slow cooling was found to be essential as rapid cooling causes
the mono-adipate co-crystal and/or 1 to precipitate regardless of the use of seeding. The
optimized process generated hemi-adipate co-crystal 19 in 90 % yield and > 99 % HPLC purity.
The final process is described in the experimental section.
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Scale-up Synthesis. The optimized methods described above were used for multi-kilogram
synthesis of 1 hemi-adipate co-crystal (19). The large-scale manufacturing process effectively
reproduced the lab experiments and generated 9.7 kg of 19 with high purity (99.4% by HPLC
A%) in excellent overall yield (80 %, table 3). In comparison, the initial medicinal chemistry
route generated 1 with 47 % yield from the same starting material 7. Gratifyingly, two silica gel
column chromatography steps in the medicinal chemistry synthesis (7 to 1) were successfully
removed in the large-scale process.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3: Overview of multi-kilogram synthesis of 1 hemi-adipate co-crystal 19
O
O
O
NH2
H
N
NHBoc
H
N
N
N
N
N
NH
O
O
O
O
H2N
adipic acid
step 3
HO
N
8
11
OH
0
.5
N
N
N
N
CDI
N
N
N
N
N
N
N
H
N
Cl
step 1
step 2
N
N
H
N
N
H
7
10
BIIB068 (1)
BIIB068 Hemi-Adipate Co-Crystal (19)
Step
Reaction
Starting
Actual
yield (kg)
7.7
Yield
Product Purity
(HPLC A %)
99.7%
weight (kg)
1
2
3
Amination
9.6
6.6
9.2
90.0%
99.0%
90.2%
Urea formation
Co-crystal
9.7
99.2%
9.7
99.4%
formation
Experimental section
Gereral. All reagents and solvents were purchased from commercial suppliers and used without
1
13
further purification. All reactions were carried out under nitrogen. H NMR and C NMR
spectra were recorded on a Bruker Avance and Varian NMR Spectrometer. Chemical shifts were
1
13
reported in ppm relative to the residual deuterated DMSO for H and C, and J values were
expressed in hertz. The following abbreviations were used to indicate multiplicities: s = singlet; d
=
doublet; t = triplet; q = quartet; m = multiplet. HPLC analysis was performed on Agilent 1290
instrument. LCMS analysis was performed on Agilent 1290 HPLC+G 6125 MS instrument.
XRPD was recorded on DX-2700BH instrument. All yields are uncorrected for purity.
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2
2
2
2
2
2
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6
Synthesis of aminopyrazole intermediate 10. To a 22 L RBF (round-bottom flask) 5.0 kg of 8
and 9.2 L (1 vol) of WFI (water for injection) were agitated at 20 °C for 1.5 hours until a
homogeneous solution was obtained. To the 200 L GL (glass-lined) reactor 9.6 kg of 7 and 15.6
kg of 2-butanol (2 vol), and 14.6 kg of WFI (2 vol) was charged and warmed at 60 °C for 30
min. The contents of the 22 L RBF were charged over 18 min, rinsing the flask with 5 kg WFI.
The reaction mixture was agitated at reflux (83-86 °C) for 19 h, at which point it was complete
by HPLC analysis (≤0.05% starting materials/intermediates). The reaction mixture was cooled to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
0 °C over 1.75 hours and 46.1 kg WFI (4.8 vol) was charged over 52 min, maintaining 35 ±
°C. A mixture of 26.2 kg of 28-30% ammonium hydroxide (7.2 equiv) in 25.7 kg WFI (3.2
vol) was charged over 1 hour 22 min maintaining 35 ± 5 °C, rinsing with 5 kg WFI. The pH of
the reaction mixture was measured to be ~11-12 via colorpHast pH paper, cooled to 20.4 °C over
4
4 min, and held at 15 ± 5 °C for 1 h. The reaction mixture was filtered through a nutsche filter,
rinsing the filter cake with 15.4 kg WFI water (1.6 vol). The filter cake (6.9”x17.5”) was loaded
onto trays and dried in a vacuum oven at 70 °C. The solids obtained constant weight after 17
1
hours to afford 7.65 kg 10 in 90 % yield. mp 145.9 °C. H NMR (400 MHz, DMSO-d6) δ 9.47
(s, 1H), 8.44 (br d, J = 4.8 Hz, 1H), 7. 91 - 7. 94 (m, 3H), 7.53 (br d, J = 8.0 Hz, 1H), 7. 22 - 7.
1
3
25 (m, 1H), 4.16 (br d, J = 6.4 Hz, 1H), 3.82 (s, 3 H), 3.75 (s, 1H), 2.35 (s, 3H). C NMR (101
MHz, DMSO-d6) δ163.87, 159.72, 158.81, 144.41, 135.71, 134.73, 129.85, 128.03, 127.35,
1
24.28, 123.40, 120.31, 106.49, 42.86, 38.68, 18.67. HRMS (ESI) calculated for C H N (M +
16 19 6
+
H) 295.1671, found 295.1653
Synthesis of 1. To a 50 L RBF was charged 22 kg (3 vol) of DMSO and 6.6 kg (22.4 mol) of
0, warming to 30 ± 5 °C to obtain a homogeneous solution. The % water was measured by KF
1
and the equivalents of CDI was adjusted starting with 1.1 equiv CDI plus water. (This KF
suggested 0.14% water which equals 2.2 mol water or 0.096 equiv water). To a 200 L GL
reactor was charged 22 kg (3 vol) of DMSO and 4.4 kg (1.198 equiv) of CDI and warm at 30 °C.
The solution of 10 in DMSO was charged to the solution of CDI in DMSO slowly over ~1h. The
flasks was rinsed with 6.3 kg of DMSO and the reaction mixture was warmed at 30 °C for 30
minutes until the reaction is complete by HPLC (≤1% 10). 3.4 kg (1.3 equiv) 3-
isopropoxyazetidine was charged and the lines were rinsed with 1 kg of DMSO. The reaction
mixture was agitated at 30 °C for 4 hours, until completion by HPLC (≤0.10% intermediate). The
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reaction mixture was cooled to 20 ± 5 °C and 23.1 kg (3.5 vol) 0 °C water was charged as
quickly as possible maintaining ≤ 30 °C. 0.132 kg (2 wt%) 1 freebase seed crystals was
immediately charged and the reaction mixture was agitated at a temperature of 20 ± 5 °C to
effect crystallization. 23.1 kg (3.5 vol) water was charged slowly over ~1h maintaining ≤ 30 °C
and age for 30 minutes at 20 ± 5 °C. Filter the reaction mixture, washing the filter cake 3 x 3 vol
water. The solid was dried in a vacuum oven at ≤ 60 °C until ≤ 2% water by KF and ≤ 3%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
DMSO by GC to afford 9.7kg (99%) 1. mp 173.7 °C. H NMR (400 MHz, DMSO-d6) δ 9.47 (s,
1
7
H), 8.45 (d, J = 5.2 Hz, 1H), 7.91 – 7.92 (m, 3H), 7.54 (br s, 1H), 7.35 (br d, J = 7.2 Hz, 1H),
.24 (d, J = 5.2 Hz, 1H), 6.85 (t, J = 5.8 Hz, 1H), 4.31 - 4.32 (m, 1H) ,4.21 – 4.22 (m, 2H), 4.01
– 4.05 (m, 2H), 3.82 (s, 3H), 3.59 – 3.63 (m, 2H), 3.54 - 3.58 (m, 1H) , 2.36 (s, 3H), 1.07 (d, J =
1
3
6
1
Hz, 6H). C NMR (101 MHz, DMSO-d6) δ163.81, 159.72, 159.63, 158.90, 141.34, 135.78,
35.17, 129.83, 128.20, 127.45, 124.25, 123.40, 120.31, 106.57, 70.26, 65.11, 57.42, 40.18,
+
40.80, 40.42, 38.73, 22.31, 18.85. HRMS (ESI) calculated for C H N O (M + H) 436.2461,
2
3
30
7
2
found 436.2427
Preparation of 1 hemi-adipate co-crystal 19. To two 50 L RBFs 36.8 L (4 vol) ethanol and
.2 kg 1 were charged, while to two 22 L RBFs 27.6L (3 vol) ethanol and 2.8 kg (0.9 equiv)
9
adipic acid were charged. The four flasks were warmed at 70-75 °C and the contents of the 22 L
flasks were transferred to the 50L slurries, rinsing with 4.6 L (0.5 vol) ethanol. The flasks were
agitated at ~72 °C until a homogenous solution was obtained, then polish filtered into a
preheated (70 °C) 200 L GL reactor, rinsing with 4.6 L (0.5 vol) ethanol. Upon reaching an
internal temperature of 70 °C, the reaction mixture was agitated 30 min to ensure a homogeneous
solution. The reaction mixture was cooled to 62 ± 2 °C and a slurry of 2% seed crystals in
saturated 1 ethanol solution was charged, rinsing the lines with 2 L of 1 hemi-adipate saturated
EtOH solution. The reaction mixture was gently agitated for 1 h at 60 ± 5 °C to effect
crystallization. The reaction mixture was cooled to 5 ± 5 °C over at least 3 h and aged at 5 ± 5 °C
for 30 min. The reaction mixture was filtered, washing 3 x 3 vol ethyl acetate, allowing each
wash to soak for 15 min. The wet 19 was dried in a vacuum oven until EtOH and EtOAc were
both ≤ 5000 ppm by GC to afford 9.67 kg (90.2 %) 1 Hemi-adipate co-crystal 19. mp 170.7 °C.
1H NMR (400 MHz, DMSO-d6) δ 11.98 (br s, 1H), 9.45 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.91 –
7
.92 (m, 3H), 7.55 (br s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 5.6 Hz, 1H), 6.83 (t, J = 5.6
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Hz, 1H) ,4.29 - 4.35 (m, 1H), 4.22 (d, J = 5.6 Hz, 2H), 4.02-4.06 (m, 2H), 3.82 (s, 3H), 3.59-3.64
(m, 2H), 3.55 - 3.58 (m, 1H), 2.36 (s, 3H), 2.18 - 2.22(m, 2H), 1.48 - 1.53 (m, 2H) ,1.08 (d, J =
1
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.4 Hz, 6H). C NMR (101 MHz, DMSO-d6) δ 174.23, 163.77, 159.70, 159.53, 158.77, 141.22,
35.71, 135.08, 129.84, 128.13, 127.46, 124.15, 123.34, 120.28, 106.50, 70.16, 65.06, 57.34,
0.76, 40.15, 38.63, 33.33, 23.97, 22.22, 18.74. HRMS (ESI) calculated for C H N O (M +
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+
H) 436.2461, found 436.2427.
AUTHOR INFORMATION
Corresponding Author
Chaomin Li  Chemical Process Development, Biogen, 225 Binney Street, Cambridge, MA,
02142, United States; orcid.org/0000-0002-1631-3955;
Email: Chaomin.li@biogen.com
Authors
Lloyd Franklin  Chemical Process Development, Biogen, 225 Binney Street, Cambridge,
MA, 02142, United States
Robbie Chen  Chemical Process Development, Biogen, 225 Binney Street, Cambridge, MA,
0
2142, United States
Tamera Mack  Chemical Process Development, Biogen, 225 Binney Street, Cambridge,
MA, 02142, United States
Michael Humora  Chemical Process Development, Biogen, 225 Binney Street, Cambridge,
MA, 02142, United States
Bin Ma  Medicinal Chemistry, Biogen, 225 Binney Street, Cambridge, MA, 02142, United
States
Brian T. Hopkins  Medicinal Chemistry, Biogen, 225 Binney Street, Cambridge, MA,
0
2142, United States
John Guzowski  Chemical Process Development, Biogen, 225 Binney Street, Cambridge,
MA, 02142, United States
Fengmei Zheng  Pharmaceutical Sciences, Biogen, 225 Binney Street, Cambridge, MA,
0
2142, United States
Michael MacPhee  Pharmaceutical Sciences, Biogen, 225 Binney Street, Cambridge, MA,
2142, United States
0
Yiqing Lin  Pharmaceutical Sciences, Biogen, 225 Binney Street, Cambridge, MA, 02142,
United States
Steven Ferguson  Chemical Process Development, Biogen, 225 Binney Street, Cambridge,
MA, 02142, United States
Daniel Patience  Chemical Process Development, Biogen, 225 Binney Street, Cambridge,
MA, 02142, United States
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George A. Moniz  CMC Management, Biogen, 225 Binney Street, Cambridge, MA, 02142,
United States
William F. Kiesman  Chemical Process Development, Biogen, 225 Binney Street,
Cambridge, MA, 02142, United States
Erin M. O’Brien  Chemical Process Development, Biogen, 225 Binney Street, Cambridge,
MA, 02142, United States
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Complete contact information is available at:
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We thank Cuicui Yuan, Deju Shang, Dong Wang, Ruiling Wang and Yinli Wu (WuXi Chemical
Service Unit) for experimental assistance, Yiqiong Guo and Miaomiao Xie (WuXi Core
Analytical Service) for analytical support.
SUPPORTING INFORMATION
The supporting information is available free of charge on the ACS publication website at DOI:
Content of supplementary material: 1) General Information; 2) Analytical Spectra of 10, 1 and
1
9; 3) Analytical Spectra of Impurities; 4) Analytical Spectra of Reaction Optimization (7 to 10,
table 1); 5) Single Crystal Structure of 19.
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1
,4-Dioxane is class 2 solvent (solvents to be limited) based on ICH Q3C with permitted daily exposure
of 3.8 mg/day.
1
0
~
0.4 % of 14 was observed for entry 11 reaction.
1
1
Initially the hemi-adipate form was named hemi-adipate salt, however, based on the calculated pKa of
.75 it is very likely that the adipate is a co-crystal. Single crystal structure of hemi-adipate was later
2
obtained and co-crystal form was therefore confirmed. see MacPhee, M.; Chen, R.; Ferguson, S.;
Franklin, L.; Mack, T. Preparation of adipate forms and compositions of biaryl inhibitors of Bruton's
tyrosine kinase. PCT Int. Appl. 2016, WO 2016201271 A1, December 15, 2016.
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carcinogenic potencies of chemical intermediates in pharmaceutical syntheses supports alternative
methods to the default TTC for calculating safe levels of impurities. Regul. Toxicol. Pharmacol. 2013, 66,
3
26–335.
1
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MacPhee, M.; Humora, M. Preparation of forms and compositions of biaryl inhibitors of Bruton's
tyrosine kinase. PCT Int. Appl. 2016, WO 2016201280 A1, December 15, 2016.
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