Characterization of a Unique Co-crystal of the BACE1 Inhibitor Verubecestat and a Reaction Intermediate: Implications for the Development of a Commercial Manufacturing Process

Article abstract of DOI:10.1021/acs.oprd.8b00015

Verubecestat is an inhibitor of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) that is currently under clinical evaluation for the treatment of prodromal Alzheimer's disease. This article describes the characterization of a co-crystal composed of a HBr salt of verubecestat and a HBr salt of a reaction intermediate as the components. This unique co-crystal was formed during production of a multikilogram batch of verubecestat. The impact of this observation is discussed, followed by the description of a modified procedure that served to prevent the formation of this highly unusual crystalline material.

Full text of DOI:10.1021/acs.oprd.8b00015

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Full Paper
Characterization of a Unique Co-Crystal of the BACE1 inhibitor
Verubecestat and a reaction intermediate: Implications to
the Development of a Commercial Manufacturing Process
Laura Artino, Richard J. Varsolona, Andrew P. J. Brunskill, William J Morris,
David A. Thaisrivongs, Jacob H Waldman, Thomas W Lyons, and Yanke Xu
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00015 • Publication Date (Web): 22 Feb 2018
Downloaded from http://pubs.acs.org on February 23, 2018
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Characterization of a Unique Co-Crystal of the BACE1 inhibitor Verubecestat and a reaction
intermediate: Implications to the Development of a Commercial Manufacturing Process
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Laura Artino,* Richard Varsolona, Andrew P. J. Brunskill, William J. Morris, David A.
Thaisrivongs, Jacob H. Waldman, Thomas W. Lyons, and Yanke Xu
Department of Process Research and Development, MRL, Merck & Co., Inc., Rahway, NJ 07065
USA
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laura_artino@merck.com
RECEIVED DATE (will be automatically inserted after manuscript is accepted).
Abstract: Verubecestat is an inhibitor of the beta-site amyloid precursor protein cleaving
enzyme 1 (BACE1) currently under clinical evaluation for the treatment prodromal Alzheimer’s
disease. This article describes the characterization of a co-crystal composed of an HBr salt of
verubecestat and a HBr salt of a reaction intermediate as the components. This unique co-crystal
was formed during a multi-kilogram production batch of verubecestat. The impact of this
observation is discussed followed by the description of a modified procedure that served to
prevent the formation of this highly unusual crystalline material.
Keywords: Co-crystal, reaction intermediate, BACE1 inhibitor, Verubecestat, Alzheimer’s
disease
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Alzheimer’s disease (AD), the most common form of dementia, is a neurodegenerative disorder
that results in the erosion of memory, vocal control, and cognitive skills before ultimately
resulting in death. There are currently greater than 5.5 million Americans living with AD in
2017, part of a worldwide total that is estimated to be 47 million.1,2 These figures are expected
to grow, with a projected patient population of over 130 million by 2050. Pharmacological
intervention is limited to treatments that temporarily address the symptoms of the disease but
there are currently no disease modifying therapies available to patients. One potential class of
therapies currently under clinical evaluation are inhibitors of the beta-site amyloid precursor
protein cleaving enzyme 1 (BACE1), which seek to interrupt the processing of amyloid precursor
protein (APP) and prevent the formation and deposition of the hallmark amyloid plaques
between neurons in AD patients.
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Verubecestat (1), an inhibitor of BACE1, is currently being evaluated in the clinic for the
treatment of prodromal Alzheimer’s disease.3,4 Our laboratories previously disclosed a
communication describing the development of a commercial manufacturing route to 1 which
relied on a reactive crystallization to form the 3-amino-1,2,4-thiadiazinene 1,1'-dioxide
heterocycle of 1.5 In our previous report, amine 2 was condensed with cyanogen bromide (CNBr)
in the absence of base to yield the HBr salt of verubecestat (1-HBr, Scheme 1). Following a salt
break with K2CO3, the active pharmaceutical ingredient (API) was isolated in 81% yield from 2.
Scheme 1: Preparation of verubecestat (1) via reactive crystallization
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The application of the reactive crystallization afforded several advantages from a processing
perspective. First, the crystallization of 1-HBr prevented the overalkylation of 1 by CNBr. (vide
infra). Second, the reactive crystallization eliminated the need to carry out an aqueous workup
and a separate isolation operation as the process afforded a direct isolation of 1-HBr and
providing the product in >99% purity. We successfully demonstrated this reactive crystallization
on an initial multi-kilogram production batch, isolating 1-HBr in 88% yield and >99 wt% purity.
Surprisingly, a second production batch yielded an atypical reaction profile. This second
production batch showed unusally high levels of cyanamide 3, formed by alkylation of 2 with
CNBr, in the supernatant after 48 hours (Figure 1).6
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Figure 1: Cyanamide intermediate 3
Furthermore, this reaction typically proceeded to 95% conversion with the balance of the starting
amine 2 in the supernatant at the end of the reaction. However, 2 was absent from the
supernatent in the second production batch, and was instead observed in the isolated solid
following filtration. Based on these observations, our initial hypothesis was that a new, poorly
soluble, phase of amine 2 or its corresponding HBr salt had crystallized under the reaction
conditions, causing the reaction progress to stall before typical conversions could be achieved.
To assess the validity of this hypothesis, the material isolated from the 2nd production batch was
rigorously characterized to confirm the composition of the isolated solid.
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Characterization of Isolated Solid from the 2nd Production Batch:
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The HBr salts of 2 and 1 were known to exist as a crystalline acetonitrile solvate and an
unsolvated phase, respectively. X-ray powder diffraction (XRPD) analysis on the solid from the
2nd production batch showed a mixture consisting of the desired phase of 1-HBr and another
unknown phase which was not similar to the 2-HBr salt acetonitrile solvate (Figure 2).
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FIGURE 2: XRPD patterns of the unknown solids from the 2nd production batch solid ([a] red),
the desired 1-HBr Salt ([b] blue) and the 2-HBr-acetonitrile solvate ([c] pink).
Scanning electron microscopy (SEM) analysis on the isolated solid (Figure 3a) indicated that two
distinct morphologies were present in the powder; smaller particles with a block-like
morphology (Figure 3b) (consistent with the previous batches of 1-HBr) and larger aggregates
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with a granule-like morphology. The observed size differential was exploited to physically
separate the larger granules from the smaller particles by sieving through a series of screen sizes.
Preliminary analysis of the smaller, block-like particles by single crystal diffraction gave unit
cell dimensions consistent with the known desired anhydrate of 1-HBr. XRPD analysis on the
larger diameter sieve cut showed that this powder had been enriched in the new phase from the
mixture with only minor amounts of 1-HBr present. Utilizing the observed differential solubility
of the unknown phase and 1-HBr, the parially purified sieved aggregates were purified further
by slurrying in acetonitrile (Figure 3c), with the resulting product essentially free of the known
anhydrous phase of 1-HBr (Figure 4). This separation/purification step significantly improved
the ability to fully characterize the unknown solid isolated from the 2nd production batch.
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(a) aggregates + primary particles (b) Small block primary particles
(c) Sieved and slurried
aggregates
(phase purified solids from 2nd
production batch)
Figure 3: SEM images of solids from second production batch showing two distinct
morphologies.
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Figure 4: XRPD comparison of unknown solid from the 2nd production batch ([a]- red), 1-HBr
([b]- blue), and the purifed granule-like solids from the second production batch reaction ([c] –
green)
To confirm the phase purity of the unknown phase, the unit cell was determined utilizing powder
indexing. The indexing solution gave a monoclinic unit cell and space group P21, with no
additional peaks that would be attributable to any other phases. The refined unit cell dimensions
are shown in Table 1 and the Pawley fit is shown in Figure 5. The expected cell volume for a
given crystal form and chemical species can be readily estimated by an atom additive approach
by considering the likely number of molecules in the crystal form and its asymmetric unit.7 The
estimated unit cell volumes for 2-HBr and 1-HBr are given in Table 2. For 2-HBr and 1-HBr
the unit cell volume for the unknown phase would require Z’ = 2 with two crystallographically
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distinct copies of the salt in the crystal form. The unit cell volume was also consistent with a
crystal form where both salts were present in the asymmetric unit. Given that the phase was now
deemed to be phase pure, the sample was evaluated by thermal analysis as well as HPLC and
ICP-MS to determine the indentity of the chemical species present.
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Table 1: Unit cell of Unknown Phase by Powder Indexing and Pawley Refinement
Crystal form
Unknown Phase
PXRD / 293K
Monoclinic, P21
7.03298
Data Type / Temperature
Crystal system, space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
γ (°)
22.60163
12.28665
90
Cell dimensions
94.56598
90
Cell Volume (Å3)
1946.85
Figure 5: Pawley Fit of XRPD Pattern for Unknown Phase
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Table 2: Estimated Cell Volumes (RT) for possible salts.
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Chemical
Species
Estimated Molecular
Volume (ų)
Formula
Z’/Z
2/4
Cell Volume (ų)
1920.2
9
2-HBr salt
C16 H19 Br1 F2 N4 O3 S1
C17 H18 Br1 F2 N5 O3 S1
C33 H37 Br2 F4 N9 O6 S2
480.1
500.6
980.7
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2/4
2002.5
1-HBr
2-HBr:1-HBr co-
1/2
1961.4
crystal
HPLC and ICP-MS revealed the purified granules were a mixture of amine 2, verbucastat 1 and
two equivalents of HBr (Table 3). This analysis, showing an approximate 1:1:2 molar mixture of
2:1:HBr, was consistent with the unknown phase that crystallized during the second production
batch being a co-crystal of the HBr salts of both 2 and the desired product 1 (Figure 6). Co-
crystals historically have been utilized in the pharmaceutical industry to improve solubility of
drug substances and typically exist between the active molecule and a co-former8. Rarer
instances exist between the molecular entity as a salt and a neutral co-former in the lattice9. The
co-crystal discussed here is unique in that it formed between a salt of a reactant and a salt of the
product.
Table 3: Component Analysis of Phase Purified Solids from the 2nd production batch.
Wt % Found in
impurity phase
Mole % of
component
Mol %
normalized to 1
Component
Amine 2
40.1
42.4
23.6
23.5
1.01
1.00
verubecestat 1
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HBr
18.6
52.8
2.25
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Figure 6: Co-crytsal from the 2nd production batch
To further support this unprecedented finding, single crystal X-ray diffraction (SC-XRD)
analysis on a sample of this purified phase was performed. The single cyrstal was obtained from
acetonitrile by slow cooling of a saturated solution.. The single crystal unit cell dimensions are
given in Table 4 along with the powder indexing cell dimensions for comparison. The cell
dimensions are the same allowing for the cell contraction during the SC-XRD experiment, which
was performed at 100K.
Table 4: Unit Cell Information for 2-HBr: 1-HBr
Crystal form
Molecular Formula
Formula Weight
2-HBr: 1-HBr
2-HBr: 1-HBr
C16H18F2N4O3S . HBr :
C17H17F2N5O3S . HBr
C16H18F2N4O3S . HBr :
C17H17F2N5O3S . HBr
955.6
955.6
Data Type / Temperature
PXRD / 293K
SC-XRD / 100K
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Crystal system, space group
Monoclinic, P21
7.0330
22.602
12.2867
90
Monoclinic, P21
6.9896
22.398
12.1743
90
a (Å)
b (Å)
c (Å)
Cell dimensions
α (°)
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β (°)
γ (°)
Cell Volume (Å3)
94.566
90
94.093
90
1946.9
1901.1
Z
(number of molecules per cell)
,
2,1
2,1
Z’ (number of molecules per
asymmetric unit)
Crystal Density (g/cm³)
1.630
1.669
SC-XRD confirmed the results of the component analysis. Figure 7a shows a thermal ellipsoid
representation of the asymmetric unit contents consisting of the protonated amine-2, the
protonated verubecestat-1 and two bromide anions. Figure 7b shows the interactions to the
bromide ions with the two organic moieties. One bromide interacts with both the protons of the
endo and exocyclic nitrogens of protonated 1, the protonated amine of 2 and the sulfonamide
proton of another crystallographically distinct molecule of 2 (Figure 7b, Br1Q). The other
bromide interacts with the amide proton of 2 and the exocyclic nitrogen proton of 1 (Figure 7b,
Br1R).
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(a) Asymmetric Unit
(b) Bromide anion interactions in 2-HBr:1-HBr
Co-crystal.
Figure 7: Single-crystal X-ray diffraction (SC-XRD) of the co-crystal formed between the HBr
salt of 2 and 1. Thermal ellipsoids are shown at the 50% probability level.
Solubility Assessment:
Single solubility measurements of the three solids (2-HBr MeCN solvate, 1-HBr, and the co-
crystal) were determined in the reaction solvent mixture at 70°C (Figure 8 and Table 5)
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Figure 8: Pictorial representation for the ternary phase diagram of 2-HBr MeCN solvate, 1-HBr,
co-crystal and reaction solvent mixture (not drawn to scale).
Table 5: Solubility Data for 2-HBr MeCN solvate, 1-HBr and the co-crystal
Phases in contact
with solvent
Solution Composition of each Solution Composition of each
Component at 25C
(mg/g solution)
Component at 75C
(mg/g solution)
2-HBr
2-HBr +
2.44
2.35
-
21.0
21.9
-
0.029
0.153
Co-Crystal-HBr
Co-Crystal-HBr
1-HBr +
0.155
0.063
0.171
1.06
0.487
0.219
0.521
2.79
Co-Crystal-HBr
1-HBr
-
1.05
-
2.58
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In order to approach full conversion, the reactive crystallization would need to be performed in
the region of the phase diagram where there is little or no co-crystal (2-HBr) out of solution. On
the phase diagram, this is the far right triangular region bound by the 1-HBr solid apex, the
ternary solution point A, and the right leg of the phase diagram. The data shows that the region
of the phase diagram where the reactive crystallization can be successfully executed is extremely
limited. Furthermore, the crystallization of the co-crystal, slows down the reaction to an
unacceptable rate providing insight to the mechanism of the reaction. The presence of cyanamide
3 in the supernatent of the second production batch coupled with the structural confirmation of
the co-crystal formation provides support that soluble HBr is critical to promote the formation of
the 3-amino-1,2,4-thiadiazinene 1,1'-dioxide heterocycle of 1 from 3. Indeed, when 3 is treated
with HBr in MeCN/IPAc, 1-HBr is readily formed.
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The low solubility of the co-crystal and the limited experimental operating range for which the
reactive crystallization is viable rendered the existing end game chemistry untenable for future
production campaigns. Our initial revision to the end game involved running the reaction under
basic conditions in order to prevent the formation of the HBr salt of 2 and subsequent co-crystal
formation. While this approach did generate 1, the cyanation of the product in the presence of
soluble organic bases such as N,N-diisopropylethylamine led to over-cyanated impurity 4
(Scheme 2) limiting the overall yield of the reaction.
Scheme 2: Conversion of 2 to 1 under basic reaction conditions
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This result led us to consider a staged approach whereby 2 would first be converted
chemoselectively to cyanamide 3 before quenching the residual CNBr and promoting
intramolecular cyclization in a subsequent operation. Towards that end, amine bases weaker than
N,N-diisopropylethylamine provided 3 as the exclusive product, but in only 50-63% conversion
(Table 6, entries 1-3). Conversions improved using inorganic bases such as KH2PO4 or NaHCO3,
which provided higher levels of 3 in a range of solvents (Table 6, entries 5, 6, 11, and 12). We
chose to optimize the reaction with NaHCO3 rather than KH2PO4, as the latter presented handling
challenges on scale due to its hygroscopicity. Some product decomposition was observed under
extended reaction times in MeOH or DMAc when using NaHCO3 (Table 6, entries 8 and 9).
However, NMP, THF and 2-Me-THF were differentiated with respect to conversion and stream
stability, and high levels of conversion were achieved using 2-MeTHF when the process was
conducted at 45 °C. After full conversion to 3, the stream was washed with aqueous sodium
thiosulfate which served to reduce residual CNBr (Scheme 3). The resulting 2-MeTHF stream
containing 3 was treated with aqueous sodium hydroxide to promote intramolecular cyclization
to 1, accompanied by only trace amounts of over-cyanated 4. Verubecestat 1 was isolated as its
para-toluenesulfonate salt (1-pTSA) for purity upgrade before performing a salt break with
potassium carbonate to afford 1 in 92% isolated yield from 2.
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Table 6: Solvent/Base Screen for the conversion of 2 to 3
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aDetermined by HPLC analysis after 24 h. bValues in parentheses refer to conversion
determined after 48 h. cReaction conducted at 45 °C.
Scheme 3: Preparation of verubecestat from 2
Conclusion:
We have described the unexpected formation and subsequent characterization of an
unprecedented crystalline intermediate identified during production scale manufacture of the
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BACE1 inhibitor verubecestat (1). Rigorous physical characterization, including single crystal x-
ray analysis confirmed the structure of this unique intermediate and enabled the design of a
revised end game chemistry to prevent its formation. This new end game relied on a
chemoselective alkylation followed by intramolecular cyclization to furnish the 3-amino-1,2,4-
thiadiazinene 1,1'-dioxide heterocycle of 1. This new end game has been demonstrated on
production scale and has provided robust access this potential disease modifying therapy.
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Author Information:
Corresponding Authors
*E-mail: laura_artino@merck.com
Notes:
The authors declare no competing financial interest.
ASSOCIATED CONTENT
The process description, NMR data, single crystal data, Pawley refinement and analytical
methods descriptions for HPLC, ICP-MS, DSC and DSC data are available in the supporting
information section. This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgements:
The authors would like to thank Xiaodong Bu, Samreen Bano, Qiang Tu, Eric Regalado and Li
Zhang
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