Process Development of the Soft Histone Deacetylate Enzyme Inhibitor SHP-141: Acylation of Methyl Paraben and Suberyl Hydroxamic Acid Formation

Article abstract of DOI:10.1021/acs.oprd.6b00280

SHP-141 (1) is a hydroxamic acid-based inhibitor of histone deacetylase enzymes which is under development for the treatment of cutaneous T-cell lymphoma. The original synthesis of 1 involved five synthetic steps beginning with suberic acid monomethyl ester. Final deprotection of the O-benzyl hydroxamate moiety using hydrogen and palladium catalyst mandated the use of metal scavengers to reduce palladium levels to within International Council for Harmonisation (ICH) guidance. Owing to the sensitivity of 1 toward self-condensation and the potential for N-O bond cleavage under hydrogenolytic conditions, we developed an alternative route to 1 which avoids Pd-mediated hydrogenation and prolonged metal scavenger treatment. This two-step process employs readily available suberic acid and methyl paraben and has successfully delivered multiple kilograms of 1 for clinical use. Importantly, crude 1 was stabilized for recrystallization in acetonitrile (ACN) solution by the addition of 0.1% citric acid and 4% water. Additionally, the filtration and drying of suitably sized aggregates of 1 with high purity (100 area%) was accomplished via temperature cycling of the 1/ACN solution.

Full text of DOI:10.1021/acs.oprd.6b00280

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Full Paper
Process Development of the Soft HDAC Inhibitor SHP-141 –
Acylation of Methyl Paraben and Suberyl Hydroxamic Acid Formation
Yijun Deng, Christina Ng DiMarco, Tanya Vakhilt, Marco Jonas, Jaclyn White, Dennis Arefyev,
Ramachandar Tokala, Parveen Akhtar, Lei Zhu, Xufeng Sun, Thomas Haimowitz, and Stephen M. Condon
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00280 • Publication Date (Web): 19 Sep 2016
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Process Development of the Soft HDAC Inhibitor
SHPꢀ141 – Acylation of Methyl Paraben and
Suberyl Hydroxamic Acid Formation
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Yijun Deng, §* Christina Ng DiMarco, § Tanya Vakhilt, § Marco Jonas, ‡ Jaclyn White, ‡
ǂ
Dennis Arefyev, ǁ Ramachandar Tokala, Parveen Akhtar, ‡ Lei Zhu, ‡ Xufeng Sun, ‡
Thomas Haimowitz§ and Stephen M. Condon§*
§TetraLogic Pharmaceuticals Corporation, 343 Phoenixville Pike, Malvern, PA 19355, United
States
‡Albany Molecular Research, Inc., 26 Corporate Circle, Albany, NY 12203, United States
ǁPatheon API Services, Inc. 101 Technology Place, Florence, SC 29501, United States
ǂAlcami, W130 N10497 Washington Drive, Germantown, WI 53022, United States
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Table of Contents Graphic:
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O
O
MsCl, DIPEA
O
CO2H
CO2H
HO2C
O
HO2C
O
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n
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Suberic Acid
Suberic Acid Poly-anhydride
O
1. Methyl Paraben, DIPEA
2. IBCF, NMM, NMP
O
OH
N
H
O
3. NH2OH HCl, NaOAc, HOAc
MeO2C
SHP-141 (1)
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ABSTRACT
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SHPꢀ141 (1) is a hydroxamic acidꢀbased inhibitor of histone deacetylate enzymes (HDACs)
which is under development for the treatment of cutaneous Tꢀcell lymphoma (CTCL). The
original synthesis of 1 involved five synthetic steps beginning with suberic acid monomethyl
ester. Final deprotection of the Oꢀbenzyl hydroxamate moiety using hydrogen and palladium
catalyst mandated the use of metal scavengers to reduce palladium levels to within ICH
guidance. Owing to the sensitivity of 1 towards selfꢀcondensation and the potential for NꢀO
bond cleavage under hydrogenolytic conditions, we developed an alternative route to 1 which
avoids Pdꢀmediated hydrogenation and prolonged metal scavenger treatment. This twoꢀstep
process employs readilyꢀavailable suberic acid and methyl paraben and has successfully
delivered multiple kilograms of 1 for clinical use. Importantly, crude 1 was stabilized for
recrystallization in acetonitrile (ACN) solution by the addition of 0.1% citric acid and 4% water.
Additionally, the filtration and drying of suitablyꢀsized aggregates of 1 with high purity (100
area%) was accomplished via temperature cycling of the 1/ACN solution.
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KEYWORDS: SHAPE, SHPꢀ141, HDAC, Suberic acid, Methyl paraben, FBRM
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INTRODUCTION
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SHPꢀ141 (1, Figure 1), a “soft” inhibitor of certain histone deacetylase enzymes or HDACs, was
invented by scientists at DanaꢀFarber Cancer Institute for the treatment of cutaneous Tꢀcell
lymphoma (CTCL).1 A topical formulation of SHPꢀ141 allows HDAC inhibition at the site of
action (i.e., skin) while the “soft” ester linkage is cleaved by plasma esterases to generate methyl
paraben (2) and suberic acid hydroxamic acid (3), thus limiting the adverse effects associated
with systemic HDAC inhibition.2 Topical SHPꢀ141 gel is currently being studied in a
randomized Phase 2 clinical trial for the treatment of earlyꢀstage CTCL.1
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[Figure 1]
The initial clinical manufacturing route is described in Scheme 1. Beginning with suberic acid
monomethyl ester (4), the acid chloride (5) was formed but not isolated. Instead, 5 was directly
reacted with Oꢀbenzyl hydroxylamine to afford the Oꢀprotected hydroxamic acid (6). LiOHꢀ
mediated saponification of the ester group and EDCꢀmediated coupling of 7 with 2 afforded
penultimate intermediate 8 which was converted to 1 following palladiumꢀcatalyzed removal of
the Oꢀbenzyl protective group.
[Scheme 1]
This first generation synthetic route presented some key challenges for commercial
manufacturing. Firstly, the final protective group removal with 10% Pd/C required prolonged
metal scavenger treatment (Smopex® 243) to reduce palladium levels to <10 ppm. Stability
studies of 1 indicated partial degradation in a variety of organic solvents (infra) thus protracted
manipulations of 1ꢀcontaining solutions were a concern. In addition, literature precedent
suggested that hydrogenation over certain palladium catalysts might afford NꢀO bond cleavage
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leading to amide formation (not shown).3 Having observed the product of NꢀO bond cleavage of
8 in our early development work, we were reluctant to rely on a chemical transformation which
might vary with scale, reaction time, and catalyst lot. Specifically, we worried that the formation
of this structurallyꢀsimilar amide (not shown) at the final step might greatly complicate the
purification of 1.4
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RESULTS AND DISCUSION
Our second generation design is outlined retrosynthetically in Figure 2. We reasoned that 1
could be prepared via the coupling of carboxylic acid 9 with hydroxylamine or a hydroxylamine
equivalent.5 Several methods are reported for the conversion of carboxylic acids (or, an
activated species derived thereof) to hydroxamic acids. Carboxylic acid 9 would, in turn, come
from the reaction of 2 with cyclic suberic anhydride (10).
[Figure 2]
Suberic anhydride (10) is reported to form when suberic acid (11) is heated with acetic
anhydride (Ac2O).6ꢀ9 In our hands, after 90 min at 150 °C, excess Ac2O was removed by
vacuum distillation and coꢀevaporation with oꢀxylene. Suberic anhydride reacted with 2 (1.1 eq)
and triethylamine (TEA, 1.1 eq) in THF (5 vol) at 20 °C to form a mixture of 9 and suberic acid
bisꢀ(methyl paraben) ester (12, Scheme 2). After 3 h, water and MTBE were added and 12 was
removed by filtration. The layers were separated and the basic aqueous phase was cooled to 0
°C, acidified with conc. HCl (pH 2ꢀ3), and filtered to provide crude 9 as a whiteꢀcolored solid.
[Scheme 2]
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The formation of 12 suggested that 9 (or its TEA salt) was further reacting with 10 to afford
linear suberyl anhydride species (not shown) that could then react with 2. High performance
liquid chromatographic (HPLC) analysis suggested that 10 was, in fact, comprised of a mixture
of suberyl polyꢀanhydrides and not a single cyclic suberyl anhydride species as shown. With the
goal of controlling suberyl polyꢀanhydride formation, we investigated other activating agents. In
the event, 11 in THF (5 vol) was reacted with TEA (1.1 eq) and activating agent (1.1 eq, Table 1)
at ꢀ5 °C. After 3 h, 2 (1.1 eq) and TEA (2.2 eq) were added and the reaction mixture was
processed as previously described. Owing to its low cost and ease of handling, methanesulfonyl
chloride (MsCl) was selected for further optimization.
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[Table 1]
Mechanistically, treatment of 11 with MsCl in the presence of TEA (1 eq) would initially form
methanesulfonyl suberate (13) and/or bisꢀmethanesulfonyl suberate (14) together with TEA⋅HCl
salt (1 eq, Scheme 3, equation 1). However, as soon as 13 (or, 14) formed, reaction with a
second equivalent of 11 (or, triethylammonium suberate) would give rise to linear suberyl
anhydride (15) (Scheme 3, equation 2). Repeating this process, i.e., 15 + 13, would afford higher
order suberyl polyꢀanhydrides (not shown). Ultimately, 11 would be entirely consumed in this
process forming a distribution of suberyl polyꢀanhydrides. Notably, any cyclic suberic anhydride
(10) which might form is subject to ring opening by 11 (Scheme 3, equation 3) or 13 (not shown)
to propagate polyꢀanhydride formation.
[Scheme 3]
Chromatographic analysis of the Stage 1a reaction mixture provided support for the formation
of suberyl polyꢀanhydrides (Figure 3A). Each of the observed peaks in the chromatogram
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represented a unique suberyl polyꢀanhydride species which were differentiated only by the
number of incorporated suberic acid monomers. No methanesulfonylꢀcontaining polyꢀsuberates
were detected by mass spectral analysis. Notably, formation of each anhydride linkage would
release one equivalent of methanesulfonic acid (MsOH, infra).
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[Figure 3]
The Stage 1b reaction of suberyl diꢀanhydride (16) with 2 under basic conditions illustrates the
formation of both 9 and 12 (Figure 4). This reaction required excess TEA (or, DIPEA) to
scavenge MsOH from the previous step (supra) as well as to deprotonate 2. Reaction of 2 at the
“internal” carbonyl group (Path A) affords an equivalent of 11 together with the methyl paraben
ester of suberyl anhydride (17) which undergoes further reaction (Path C) to provide two
equivalents of 9. Alternatively, 17 can react with 2 to generate 11 plus 12 (Path D). When 2
reacts at the “external” carbonyl group of 16, both 9 and 15 are formed (Path B); 15 would
further react to provide 9 and 11 (not shown). Thus, this mechanistic interpretation suggests that
9 will be formed in no greater than 50% yield and that both 11 and 12 will be byꢀproducts in the
reaction. Because of the low cost and ready availability of 11 and 2, we reasoned that this
limitation on the theoretical yield of 9 would be acceptable on commercial scale.
[Figure 4]
This twoꢀstep process requires sufficient base to neutralize both the HCl (from MsCl) and
MsOH (via anhydride formation), i.e., twice the equivalents of MsCl used in Step 1a, plus the
amount of base required to deprotonate 2 used in Step 1b. We performed a series of experiments
to evaluate the required quantity and order of addition of reagents (Table 2). Comparable yields
of isolated 9 were observed when the first charge of organic base (DIPEA) was added in a single
portion (Table 2, Entry 1) or divided between Step 1a and Step 1b (Table 2, Entry 2). However,
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when DIPEA (2 eq) was added in one portion on 40 gꢀscale (i.e., suberic acid), the
heterogeneous reaction mixture became too thick to stir owing to precipitation of both
DIPEA⋅HCl and DIPEA⋅MsOH salts (Table 2, Entry 3). Employing an initial charge of DIPEA
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(1.3 eq), the reaction mixture was warmed to 50 °C following 2/DIPEA (1.75 eq) addition (Table
2, Entry 4). After ca. 16 h (i.e., overnight) at 50 °C and standard workꢀup, 9 was isolated in 42%
yield.
[Table 2]
Recognizing that the addition of solid 2 and the slow filtration of 12 might present problems
during commercial manufacturing, we added 2 as a THF (2 vol) solution and developed an
alternative workꢀup procedure for 9 (Table 2, Entry 5). To this end, when the reaction was
deemed complete, the reaction mixture (Step 1b) was diluted with toluene (5 vol), water (10 vol)
and DIPEA (1.0 eq) at 50 °C. After 20 min, the layers were separated and the warm toluene
layer was extracted with DIPEA/water to remove additional 9. The combined aqueous phase
was then cooled and acidified (pH 2ꢀ3) with conc. HCl. The resultant solid was collected by
filtration and processed as before to provide 9 in 37% yield. This process was successfully
demonstrated beginning with 7.5 kg of 11 (Table 2, Entry 6). Residual 11 and 2 were
conveniently removed by suspending crude 9 in 1:1 MeOH/water at ambient temperature.
Following filtration, the wet cake was washed with 1:1 MeOH/water and dried in vacuo for 12 h
at 35ꢀ45 °C to provide 5.2 kg (39%, 100 area%) of 9 as a whiteꢀcolored solid.
To establish the reaction time course, we monitored the formation of 9 and 12 at 50 °C by
HPLC analysis. As shown in Figure 3B, the reaction was complete between 5 and 14 h.
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Interestingly, the rates of formation for both 9 and 12 were nearly superimposable which
suggested a common reaction pathway.
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For the preparation of 1, we activated 9 using isobutylchloroformate (IBCF) in the presence of
Nꢀmethylmorpholine (NMM) followed by the addition of 50% aqueous NH2OH (Scheme 4). We
chose 1ꢀmethylꢀ2ꢀpyrrolidinone (NMP) as solvent owing to its miscibility with water. Both the
activation of 9 with IBCF/NMM (to afford 18) and the addition of aqueous NH2OH to 18/NMP
were exothermic reactions and thus required low temperature, i.e., ≤ ꢀ10 °C, and efficient
mixing. SHPꢀ141 (1) was partly unstable under these conditions giving rise to oligomeric
species 19 and 20 as prominent degradants – the former being the product of 1 selfꢀcondensation
(with loss of 2) while the latter partly arose via the reaction of 1 with unreacted 18. The rate of
formation of 19 and 20 accelerated with increasing temperature and concentration of 1. When
minimal agitation was applied, the symmetric anhydride (21) was observed in >10 area% by
HPLC analysis.
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[Scheme 4]
Not surprisingly, 1 was unstable in the presence of excess NH2OH owing to sensitivity of the
Oꢀacylated methyl paraben moiety. In addition, 1 degraded under strongly acidic conditions (pH
<3). We observed, however, that slightly acidic (pH 4ꢀ6) solutions of 1 had improved stability.
We therefore attempted to maintain the reaction pH at 5.5ꢀ6.5 throughout the addition of aqueous
NH2OH/NMP by employing acetic acid as a buffering agent.
To this end, a preꢀcooled (<10 °C) solution of 50% aqueous NH2OH (1.05 eq) and HOAc (2.0
eq) in NMP (1 vol based on 9) was added slowly to a cooled (ꢀ20 °C) solution of 18/NMP [NB:
prepared by adding IBCF (1.1 eq) and NMM (1.1 eq) to 9 (20 g, 1.0 eq) in NMP (5 vol) at ꢀ20
°C.] The reaction mixture was warmed to 10 °C. After 16 h, the product (1) was precipitated by
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the addition of water (15 vol) then collected by filtration, washed with water, and airꢀdried
(Table 3, Entry 1). To facilitate a final, polish filtration, crude 1 was dissolved in THF
containing 1% HOAc (5 vol), filtered and then reꢀprecipitated by the addition of water (20 vol).
The solid was collected by filtration, washed with water and dried in vacuo at 40 °C to afford
17.0 g (81%) of 1 as whiteꢀcolored needles (92.1 area%; 20: 7.0 area%).
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To minimize adventitious formation of 20, we reasoned that the inverse addition of 18/NMP to
the NH2OH/water/NMP solution would result in less 18 being available to react with 1.
Application of the inverse addition resulted in reduced levels of 20 compared to the control
conditions (5.3 area% vs. 0.76 area%) when analyzed prior to the first water addition (Table 3,
Entries 1 and 2) and afforded 1 in good yield with excellent chemical purity (Table 3, Entry 2).
Notably, in the two aforementioned reactions, the amount of 20 increased to 7.0 area% and 1.4
area%, respectively, after the precipitated 1 was allowed to stir overnight prior to final filtration.
We further modified the process by employing NH2OH·HCl as the hydroxylamine source (Table
3, Entry 3). Differential scanning calorimetric (DSC) analysis of 50% aqueous NH2OH and
buffered (NaOAc/HOAc) NH2OH·HCl solutions showed a higher onset temperature (115 °C vs.
102 °C, respectively) and lower decomposition energy (ꢀH = ꢀ66 vs. ꢀ1654 J/g, respectively) for
the latter condition.
[Table 3]
As described in Figure 5A, four acetateꢀbased buffers were prepared (pH range: 5.1 to 6.1),
combined with NH2OH·HCl, and independently evaluated using the standard Stage 2b reaction
conditions. Notably, the pH 5.1 buffer was exhausted at ca. 60ꢀ70% addition of 18/NMP as
evidenced by the drop in pH (Figure 5B, Entry 1). Since free NH2OH has a pKa of 5.95, this
drop in pH likely resulted in less free NH2OH being available for reaction with 18 (infra). The
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remaining three buffer systems maintained a constant pH throughout the addition of the 18/NMP
solution (Figure 5B, Entries 2ꢀ4).
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[Figure 5]
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We monitored the four reaction profiles by HPLC analysis. Following the standard protocol
(Stage 2a), 18 was formed with high chemical purity in each of the four reactions (Table 4).
These results demonstrated that when reaction pH was maintained at 5.5ꢀ6.5, 1 was afforded in
≥90 area% chemical purity while minimizing the formation of 20.
[Table 4]
The final solid form of 1 is not pharmaceutically important as 1 is dissolved then formulated as
a gelled solution for topical application. As described previously, the addition of water to the
Stage 2b reaction mixture afforded solid 1 as fine particles which presented problems during
filtration, i.e., slow filtration rate. Thus, to help with this final processing step, we investigated
alternative polymorphic forms of 1 in order to identify a more easily filterable solid form.
A set of 13 organic solvents was investigated for the recrystallization of 1 using both slow
cooling (20 °C/h) and fast cooling (4 °C) procedures (Supplemental Table S1); the dried, isolated
solids were then examined by Xꢀray powder diffraction (XRPD). Previously, we observed that 1
was unstable in acetonitrile (ACN) and other polar organic solvents for ≥4 h at >45 °C but could
be partly stabilized with <1% HOAc or citric acid (Supplemental Table S2). Thus, for this
investigation, we added 0.2% HOAc (v/v) to the solvents in order to stabilize these 1ꢀcontaining
solutions. From this analysis, we identified two unique polymorphic forms of 1 (Table 5). The
XRPD patterns of 1 Form A and 1 Form B are shown in Supplemental Figure S1. Microscopic
analysis of 1 Form A revealed birefringent aggregates while 1 Form B existed predominantly as
needleꢀshaped crystals (Supplemental Figure S2). Disappointingly, all attempts to generate
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larger crystals of either 1 Form A or 1 Form B were unsuccessful (data not shown). We
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[Table 5]
We compared the combination of isopropyl acetate (IPAc), methyl tertꢀbutyl ether (MTBE) or
toluene with water for the Stage 2b precipitation process. A mixture of purified 1 (10 g, ≥95
area%) in NMP (8.5 vol), water (1.1 vol), and HOAc (1 vol) was cooled to 0 °C. The described
coꢀsolvent (4 vol) was added either before or beforeꢀandꢀafter the addition of water (20 vol)
causing the precipitation of 1 (Table 6). The precipitated 1 was collected by filtration, washed
with water, MTBE, airꢀdried, then analyzed by HPLC. The recovery of 1 from all the
experiments was ca. 90%. When toluene was employed as coꢀsolvent, peaꢀsized aggregates of 1
formed within a toluene/water/NMP emulsion which allowed an improved filtration rate of 1
compared to material precipitated from water only (ca. 1 h on 25 gꢀscale), IPAc/water or
MTBE/water processes (Table 6, Entries 1ꢀ5). On larger scale, however, a slower filtration rate
was observed perhaps due to disaggregation of 1 during the MTBE washes resulting in a more
compressed filter cake (Table 6, Entries 6ꢀ8). When preꢀcooled (0ꢀ8 °C) 12% NaCl/water (brine)
was used in combination with toluene, a complete aqueous/organic phase separation was
observed and 1 was obtained as an easilyꢀfilterable granular solid (Table 6, Entry 9). A
comparison of preꢀcooled 8%, 10%, and 12% brine suggested that the most granular form of 1
was obtained using toluene and 8% brine (data not shown). Importantly, the application of
toluene and 8% brine exhibited reduced degradation of 1 over time as compared to IPAc/water
(Supplemental Table S3).
[Table 6]
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Owing to the temperature sensitivity of 1ꢀcontaining solutions, initial attempts to purify 1
focused on low temperature (<40 °C) trituration of crude 1 in organic solvents such as DCM or
MTBE. Although these processes were effective at removing 20 and other related impurities,
they were less able to purge oligomeric species 19. To develop a more effective purification
process, we explored the solvent systems first identified in the polymorphic characterization of 1
(Supplemental Tables S1 and S2). For purification at Stage 3, crude 1 (5 g) was added to ACN
containing 0.1% wt/v citric acid (12ꢀ15 vol) with or without water and the heterogeneous
mixture was warmed to 50 °C. After 2 h, the clear solution was cooled to ambient temperature
and maintained for 16 h. The precipitated solid was collected by filtration, washed with ACN (2
× 5 vol) and dried in vacuo at 40 ± 5 °C. Analyses (HPLC) of the collected solids and filtrates
are presented in Table 7.
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[Table 7]
These results suggested that higher percentages of water in ACN, although providing material
with high purity, could negatively impact the recovery of 1 (Table 7, Entry 3). Gratifyingly,
however, there was no impact on the purity or recovery of 1 when the heating time was extended
to 6 h (Table 7, Entry 4). Based on these observations, crude 1 (44 g, 91.4 area%) was treated
with 0.1% citric acid/4% water in ACN (12 vol) at 45 °C to provide purified 1 (52% recovery,
99.6 area%). Despite the modest recovery for this process (50ꢀ70%), the excellent purity and
solution stability of 1 allowed us to explore the final isolation conditions.
To optimize the particle size of purified 1 in advance of filtration, we turned to Focused Beam
Reflectance Measurement (FBRM) technology to understand how particle size might vary across
temperature cycles. Acetonitrile containing 1, 0.1% citric acid and 4% water were placed into a
reactor containing an FBRM probe (FBRM® D600L, MettlerꢀToledo) and the heterogeneous
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mixture was stabilized at 23 °C. The mixture was then subjected to four temperature cycles
while monitoring particle size distribution (Table 8). The data from Cycles 2 and 3 suggested
that larger particles might arise via a more rapid cooling phase (Supplemental Figure S3).
Application of a modified temperature cycle (see: Experimental) consistently provided 1 Form A
in >99 area% purity by HPLC analysis. In order to improve the recovery of 1, we lowered the
final isolation temperature to 10 °C. On 10 gꢀscale, this modification afforded a 72% recovery of
1 Form A (100 area%).
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[Table 8]
CONCLUSIONS
In summary, a robust three stage process was developed for the synthesis and purification of 1
from inexpensive starting materials (Scheme 5). We successfully demonstrated the chemistry
and process on kgꢀscale and applied this process for the cGMP preparation of >10 kg of 1 for
clinical use. Key modifications include: i. the implementation of a hot toluene extraction to
separate 12 from 9·DIPEA salt (Stage 1b); ii. the inverse addition of 18 to a buffered (pH 5ꢀ6)
hydroxylamine solution, i.e., NH2OH·HCl (1.3 eq)/NaOAc (1.3 eq)/HOAc (5.6 eq, Stage 2b); iii.
the precipitation of 1 from the Stage 2b reaction mixture using preꢀcooled 8% brine/toluene to
increase the particle size and improve the filtration rate of crude 1; iv. the recrystallization of
crude 1 using 4% water in ACN containing 0.1% citric acid (Stage 3); and, v. the incorporation
of temperature cycling at Stage 3 to generate 1 Form A of sufficient size to facilitate final
isolation at 10 °C.
[Scheme 5]
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13
General. H and C NMR spectra were obtained at 500 and 125 MHz, respectively, using a
Bruker Avance spectrometer using tetramethylsilane as the internal standard. Reactions were
routinely performed under a nitrogen atmosphere using standard glassware and high purity,
commercialꢀgrade solvents. LC/MS analysis was performed on a ThermoꢀFisher MSQ Plus
instrument with a Gemini 5 ꢁ C6ꢀPhenyl 110 Å column (50 × 4.60 mm) using standard gradient
conditions (A: water containing 0.1% HOAc v/v; B: acetonitrile containing 0.1% HOAc v/v);
MS data were acquired with ESI, positive ionization; UV detection at 254 nm unless otherwise
noted. Compound purifications were performed using either flash silica gel chromatography or
preparative HPLC (Varian Prostar) in normal phase (SiO2, EtOAc/hexane, 250 × 41.4 mm) or
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reversedꢀphase (C18, 100 Å, 60 ꢁ, 250 × 41.4 mm). Abbreviations: ACN, acetonitrile; CDCl3,
chloroformꢀd; DIPEA, diisopropylethylamine; DMSO, dimethylsulfoxide; eq, equivalents; g,
grams; HCl, hydrochloric acid; h, hours; HOAc, acetic acid; IBCF, isobutyl chloroformate; kg,
kilograms; min, minutes; MeOH, methanol; MHz, megahertz; mL, milliliters; mol, moles; MS,
mass spectrum; MsCl, methanesulfonyl chloride; MTBE, methyl tertꢀbutyl ether; NaOAc,
sodium acetate; NMM, Nꢀmethylmorpholine; NMP, 1ꢀmethylꢀ2ꢀpyrrolidinone; NMR, nuclear
magnetic resonance; ppm, parts per million; THF, tetrahydrofuran; vol, volumes.
Octanedioic acid monoꢀ(4ꢀmethoxycarbonylꢀphenyl) ester (9). To a solution of Suberic Acid
(7.5 kg, 43.0 mol) in THF (30 L, 4.0 vol) as added methanesulfonyl chloride (4.9 kg, 1.0 eq) at
15ꢀ20 °C followed by DIPEA (7.2 kg, 1.3 eq). The resulting light yellowꢀcolored solution was
agitated for 1 h at 15ꢀ25 °C. Methyl Paraben (6.9 kg, 1.05 eq) in THF (15 L, 2 vol) was added at
15ꢀ25 °C followed by a second portion of DIPEA (9.7 kg, 1.75 eq). The reaction mixture was
then heated to 50 °C. After 20 h, a third portion of DIPEA (5.6 kg, 1.0 eq) was added followed
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by the addition of toluene (37.5 L, 5 vol) and water (75.0 L, 10 vol). After 20 min, agitation was
stopped and the layers were separated. The organic layer was washed once with water (37.5 L, 5
vol) containing DIPEA (2.8 kg, 0.5 eq). The combined aqueous layers were cooled to 15ꢀ25 °C
and the pH was adjusted to 2.5 using conc. HCl (~7 L) which resulted in the precipitation of 9 as
a whiteꢀcolored solid. The thick suspension of 9 was filtered through standard weave,
polypropylene cloth and washed three times with water (37.5 L, 5 vol). The wet cake was
conditioned overnight and 1:1 MeOH:Water (42 L, 5.6 vol) was added. After stirring for 1 h,
the suspension was filtered through standard weave, polypropylene cloth, washed three times
with 1:1 MeOH:Water (21 L, 2.8 vol) then dried in vacuo at 35ꢀ45 °C to afford 5.2 kg (39%) of 9
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1
as a whiteꢀcolored solid (98.8 area%). H NMR (500 MHz, DMSOꢀd6): δ 11.98 (br s, 1H), 8.01
(d, J = 8.75 Hz, 2H), 7.28 (d, J = 8.75 Hz, 2H), 3.86 (s, 3H), 2.60 (t, J = 7.35 Hz, 2H), 2.21 (t, J
= 7.35 Hz, 2H), 1.64 (app quint, J = 7.30 Hz, 2H), 1.52 (app quint, J = 7.30 Hz, 2H), 1.34 (m,
13
4H) ppm; C NMR (125 MHz, DMSOꢀd6): δ 174.3, 171.3, 165.4, 154.1, 130.6, 127.0, 122.1,
52.1, 33.5, 33.3, 28.1, 27.9, 24.2, 24.0 ppm. HRMS (ESI), m/z 309.1187 [(M+H), calcd for
C16H21O6: 309.1338].
4ꢀ(7ꢀHydroxycarbamoylꢀheptanoyloxy)ꢀbenzoic acid methyl ester (1). To a solution of
NH2OH⋅HCl (0.70 kg, 1.25 eq), water (2.5 L, 1 vol) and HOAc (2.71 kg, 5.6 eq) was added
NaOAc (0.87 kg, 1.30 eq) and NMP (9.38 L, 3.75 vol). The heterogeneous mixture (i.e., thin
slurry) was cooled to ca. ꢀ10 °C and stirred overnight.
To a solution containing 9 (2.50 kg, 1 eq) in NMP (10 L, 4 vol) at ꢀ3 °C was added IBCF (1.22
kg, 1.10 eq) followed by NMM (0.86 kg, 1.05 eq). The resultant suspension of 17/NMP was
added to the preꢀformed hydroxylamine solution over 55 min at ca. ꢀ10 °C. Toluene (10 L, 4 vol)
was added to the reaction mixture followed by the addition over 70 min of preꢀcooled (~6 °C),
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8% NaCl/water (37 L, 15 vol). After 30 min, the solid was collected by filtration through
standard weave, polypropylene cloth and washed with water (3 × 8.5 L, 3 × 3.4) then MTBE (3 ×
8.5 L, 3 × 3.4 vol). The wet cake was conditioned overnight then dried in vacuo at room
temperature to provide 1.97 kg (75%) of 1 as a whiteꢀcolored solid (99.8 area%).
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A solution containing crude 1 (1.97 kg) and citric acid (26 g, 0.11% based on ACN) in 4%
water/ACN (25.6 L, 13 vol) was heated to 45 °C. The slightly hazy solution was filtered through
a 1.2 ꢁm filter cartridge and the filtrate was warmed to 45 °C to dissolve any precipitated solid
then cooled to 30 °C over 30 min. Upon reaching 30 °C, the solution was warmed to 35 °C and
maintained for 30 min. The solution was cooled to 10 °C over 6 h and the resultant suspension
was stirred at 10 °C. After 16 h, the solids were collected by filtration, washed three times with
ACN (3 × 2 vol ACN) and dried in vacuo at room temperature to provide 1 Form A (1.49 kg,
1
76% recovery) as a whiteꢀcolored solid (100 area%). H NMR (500 MHz, DMSOꢀd6): δ 10.34
(s, 1H), 8.67 (s, 1H), 8.02 (d, J = 8.75 Hz, 2H), 7.29 (d, J = 8.75 Hz, 2H), 3.86 (s, 3H), 2.60 (t, J
= 7.40 Hz, 2H), 1.96 (t, J = 7.40 Hz, 2H), 1.64 (m, 2H), 1.51 (m, 2H), 1.36 (m, 2H), 1.28 (m, 2H)
13
ppm; C NMR (125 MHz, DMSOꢀd6): δ 171.4, 169.1, 165.5, 154.2, 130.7, 127.1, 122.2, 52.2,
33.4, 32.2, 28.2, 28.0, 24.9, 24.1 ppm. HRMS (ESI), m/z 324.0960 [(M+H), calcd for
C16H22NO6: 324.1447].
ASSOCIATED CONTENT
SUPPORTING INFORMATION
The supporting information contains the chemical characterization of 12, 19, and 20. This
material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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*Corresponding Authors:
7
8
9
Phone: 001ꢀ610ꢀ889ꢀ9900, Fax: 001ꢀ610ꢀ889ꢀ9994. Eꢀmail: Yijun.Deng@tetralogicpharma.com
and Stephen.Condon@tetralogicpharma.com.
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Present Addresses
Yijun Deng, VenatoRx Pharmceuticals, 30 Spring Mill Drive, Malvern, PA 19355 (USA);
Thomas Haimowitz, Fox Chase Chemical Diversity Center, Inc., 3805 Old Easton Road
Doylestown, PA 18902 (USA).
Author Contributions
All authors contributed to the writing or editing of this manuscript. All authors contributed to
the design, synthesis, or analysis of the chemistry described in this manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank their colleagues at TetraLogic Pharmaceuticals Corporation, Cambridge Major
Laboratories (Alcami), IRIX Pharmaceuticals (Patheon), and Albany Molecular Research, Inc.
for their helpful advice and support.
ABBREVIATIONS USED
2ꢀMeTHF, 2ꢀmethyltetrahydrofuran; ACN, acetonitrile; Ac2O, acetic anhydride; CDCl3,
chloroformꢀd; CTCL, cutaneous Tꢀcell lymphoma; DCM, dichloromethane; DIPEA,
diisopropylethylamine; DMSO, dimethylsulfoxide; DSC, differential scanning calorimetry;
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DVS, dynamic vapor sorption; EDC, 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide; EtOAc,
ethyl acetate; EtOH, ethanol; eq, equivalents; FBRM, focused beam reflectance measurement; g,
grams; HCl, hydrochloric acid; HDAC, histone deacetylase enzyme; h, hours; HOAc, acetic
acid; HPLC, high performance liquid chromatography; IBCF, isobutyl chloroformate; iꢀPrOH,
isopropanol; IPAc, isopropyl acetate; kg, kilograms; LiOH, lithium hydroxide; min, minutes;
MEK, methyl ethyl ketone; MeOH, methanol; MHz, megahertz; MIBK, methyl isobutyl ketone;
mL, milliliters; mol, moles; MS, mass spectrum; MsCl, methanesulfonyl chloride; MsOH,
methanesulfonic acid; MTBE, methyl tertꢀbutyl ether; NaOAc, sodium acetate; nꢀBuOH, normal
butanol; ND, not detected; NMM, Nꢀmethylmorpholine; NMP, 1ꢀmethylꢀ2ꢀpyrrolidinone; NMR,
nuclear magnetic resonance; Pd/C, palladium on carbon catalyst; ppm, parts per million; RH,
relative humidity; RM, reaction mixture; THF, tetrahydrofuran; TEA, triethylamine; vol,
volumes; v/v, volumeꢀtoꢀvolume; wt/v, weightꢀtoꢀvolume; XRPD, Xꢀray powder diffraction
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REFERENCES
(1)
Guitart, J.; Bradner, J. E.; Landau, S. B. J. Clin. Oncol. 2014, 32:5s (suppl; abstr 8525).
(2) Ali, A.; Bluteau, O.; Messaoudi, K.; Palazzo, A.; Boukour, S.; Lordier, L.; Lecluse, Y.;
Kim, Y. H.; Krathen, M.; Duvic, M.; Wong, H.; Porcu, P.; Tacastacas, J.; Korman, N. J.;
Rameau, P.; KrausꢀBerthier, L.; JacquetꢀBescond, A.; Lelièvre, H.; Depil, S.; Dessen, P.; Solary,
E.; Raslova, H.; Vainchenker, W.; Plo, I.; Debili, N. Cell Death Disease 2013, 4, e738; doi:
10.1038/cddis.2013.260.
(3)
Nikam, S. S.; Kornberg, B. E.; Johnson, D. R.; Doherty, A. M. Tetrahedron Lett. 1995,
36, 197ꢀ200.
(4)
Koch, G.; Kottirsch, G.; Wietfeld, B.; Küsters, E. Org. Process Res. Dev. 2002, 6, 652ꢀ
659.
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(5)
Hazeldine, S. T.; Polin, L.; Kushner, J.; Paluch, J.; White, K.; Edelstein, M.; Palomino,
5
6
7
8
E.; Corbett, T. H.; Horwitz, J. P. J. Med. Chem. 2001, 44, 1758ꢀ1776.
(6) Botta, C. B.; Cabri, W.; Cini, E.; De Cesare, L.; Fattorusso, C.; Giannini, G.; Persico, M.;
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Petrella, A.; Rondinelli, F.; Rodriquez, M.; Russo, A.; Taddei, M. J. Med. Chem. 2011, 54, 2165ꢀ
2182.
(7)
(8)
Mai, A.; Esposito, M.; Sbardella, G.; Massa, S. OPPI Briefs 2001, 33, 391ꢀ394.
Gediya, L. K.; Chopra, P.; Purushottamachar, P.; Maheshwari, N.; Njar, V. C. O. J. Med.
Chem. 2005, 48, 5047ꢀ5051.
(9)
Oger, F.; Lecorgne, A.; Sala, E.; Nardese, V.; Demay, F.; Chevance, S.; Desravines, D.
C.; Aleksandrova, N.; Le Guével, R.; Lorenzi, S.; Becarri, A. R.; Barath, P.; Hart, D. J.; Bondon,
A.; Carettoni, D.; Simonneaux, G. Salbert, G. J. Med. Chem. 2010, 53, 1937ꢀ1950.
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O
O
OH
O
OH
N
OH
HO
plasma
H
+
N
8
9
O
H
MeO2C
MeO2C
O
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SHP-141 (1)
Methyl Paraben (2)
Suberic Acid Hydroxamic Acid (3)
Figure 1. Enzymatic deactivation of SHPꢀ141 (1) by plasma esterases
Scheme 1. First Generation Synthesis of SHPꢀ141 (1)
O
O
O-benzyl hydroxylamine
oxalyl chloride
MeO
MeO
LiOH
OH
Cl
O
O
Suberic Acid monomethyl ester (4)
5
O
O
O
O
MeO
HO
N
N
H
H
O
O
O
6
7
O
O
H2, 10% Pd/C
N
2, EDC
SHP-141 (1)
H
O
MeO2C
8
O
O
OH
O
O
N
OH
H
O
O
MeO2C
MeO2C
SHP-141 (1)
9
O
OH
O
+
MeO2C
Methyl Paraben (2)
O
Suberic Anhydride
(10)
Figure 2. Retrosynthetic Analysis of 1. The Second Generation Approach
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Scheme 2. The Reaction of Methyl Paraben (2) with Suberic Anhydride (10)
6
7
8
9
O
Ac2O, reflux
2, TEA, THF
16 h
CO2H
O
HO2C
(37%)
(quant.)
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O
Suberic Acid (11)
10
CO2Me
O
O
O
O
O
OH
+
O
O
MeO2C
MeO2C
9
12
Table 1. The Synthesis of 9 using Selected Activating Agents
Activating
Agent
9
Entry
(Isolated Yield, %)
MsCl
Ms2O
Ac2O
37
39
23
27
1
2
3
4
Me3CCOCl
Scheme 3. Stage 1a: The Formation of Linear Suberyl Anhydride and Polyꢀanhydrides
MsCl (1 eq),
TEA (1 eq)
CO2H
CO2H
CO2Ms
CO2Ms
.
(1)
(2)
TEA HCl
+
+
HO2C
HO2C
HO2C
MsO2C
HO2C
11
11
13
14
O
O
CO2Ms
HO2C
O
CO2H
+
13
15
O
O
O
(3)
CO2H
+
O
HO2C
O
CO2H
HO2C
11
15
O
10
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Figure 3. HPLC Analysis of the Stage 1a Reaction Mixture (λ = 210 nm) (A); and, the
Formation of 9 and 12 during Stage 1b (B)
O
O
O
CO2H
HO2C
O
O
O
16
Path A
Path B
OH
HO
2
2
MeO2C
CO2Me
11 +
O
O
O
9 + 15
HO2C
O
O
Path C
Path D
CO2Me
17
OH
HO
2
2
MeO2C
CO2Me
9 (2 equivs)
11 + 12
Figure 4. Stage 1b: The Reaction of 2 with Suberyl Polyꢀanhydrides
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Table 2. Results for the Formation of 9 by Varying the Quantity of Base Added at Stage 1a
and Stage 1b
7
8
9
Step 1a:
11 + MsCl
Step 1b:
Suberyl Anhydrides + 2
Total
DIPEA
(eq)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11,
g
Yield,1
%
Entry
MsCl
DIPEA
(eq)
2.1
2
(eq)
1.1
1.1
1.1
1.05
1.05
1.05
DIPEA
(eq)
1.1
(eq)
1.0
1.0
1.0
1.0
1.0
1.0
20
10
40
40
70
3.2
3.1
na
3.05
3.05
3.05
40
42
1
2
3
4
5
6
1.3
2.0
1.3
1.3
1.8
na
1.75
1.75
1.75
na2
423
393,4
393,4,5
7500
1.3
General procedure: Suberic acid (11, 1 eq) was dissolved in anhydrous THF (5 vol) containing DIPEA. The
mixture was cooled to 0 °C and MsCl was added. After 1 h, solid 2 and DIPEA were added and the mixture was
warmed to ambient temperature. After 16 h, water (20 vol), MTBE (5 vol), and DIPEA (1.0 eq) were added. After
30ꢀ60 min, 12 was removed by vacuum filtration. The biphasic filtrate was separated and the 9ꢀcontaining aqueous
layer was washed with MTBE (5 vol), cooled to 0 °C, and acidified (pH 2ꢀ3) with conc. HCl. Crude 9 was collected
by filtration then suspended in MeOH (2 vol) for 1 h at ambient temperature. Water (2 vol) was added and the
heterogeneous mixture was stirred for 1 h and filtered. The wet cake was washed with 1:1 MeOH/water and dried
under vacuum at 50 °C to afford 9 as a whiteꢀcolored solid. Yield of 9 based on 11. Reaction mixture was too
thick to stir at Step 1a; therefore, the reaction was discontinued. 3 Following 2 addition, reaction mixture heated to
50 °C for 16 h. 4 Methyl Paraben (2) added as a THF (2 vol) solution. Modified workꢀup using toluene extraction
of 12 (supra). 5 Crude 9 triturated in 1:1 MeOH/water (supra). na, not applicable.
1
2
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Scheme 4. Stage 2: The Formation of 18 and Its Reaction with 50% Aqueous NH2OH
Containing HOAc
7
8
9
Stage 2b:
50% aq. NH2OH,
HOAc, NMP, -20 ο
Stage 2a:
O
O
O
IBCF, NMM,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NMP, -10 ο
C
O
O
C
OH
O
O
O
O
O
MeO2C
MeO2C
9
18
O
O
OH
OH
O
O
O
N
H
N
N
+
H
H
O
O
O
MeO2C
MeO2C
1
19
CO2Me
O
O
O
O
O
+
N
H
O
O
MeO2C
20
O
O
O
O
O
+
O
O
CO2Me
MeO2C
21
Table 3. A Comparison of the Effects of Acetic Acid or Acetate Buffer on Stage 2b
NH2OH
Source
(eq)
HPLC
Analysis2
(area%)
Yield
(Purity,
area%)
9
IBCF NMM
HOAc NaOAc
(eq)
Entry
(g) (eq)
(eq)
(eq)
Aq.
HCl
1
20
81% (92.1)3
11
2
3
20
20
20
1.1
1.1
1.1
1.1
1.1
1.1
1.05
1.2
0
0
0
2.0
2.2
1.2
0
0
91.6
5.3
91.7 0.76 73% (98.6)4
1.2
1.2
94.7 ND 75% (99.2)5
General procedure: Carboxylic acid 9 (20 g, 1 eq) was dissolved in anhydrous NMP (5 vol). The solution was
cooled to ꢀ5 °C and IBCF and NMM were added. After 0.5 h, the thin suspension was transferred to an addition
funnel (18: >95 area%). The reactor was charged with the described NH2OH source and NMP (1.1 vol based on 9).
After cooling to ꢀ10 °C, the 18/NMP solution was added over 24 min then the reaction mixture was warmed to 10
°C. After 16 h, water (19 vol) was added and the resultant solid was collected by filtration, washed (water) and airꢀ
dried. Crude 1 was dissolved in THF containing 1% HOAc (5 vol) to facilitate a polish filtration step. Water (20
vol) was added over 1 h and the resulting suspension was aged (0.33 h), filtered, washed with water, and dried to
1
constant weight under vacuum at 40 °C to afford 1 (15.3 g) as a white, needleꢀlike solid. Addition of
hydroxylamine to 18/NMP; see text. 2 HPLC analyses performed prior to first water addition. 3 20: 7.0 area%. 4 20:
1.4 area%. 5 20: 0.4 area%. ND, not detected.
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Figure 5. The pH of Several Prepared Hydroxylamine/Acetate Solutions (A); and, the
Effect on pH when Adding 18/NMP to Hydroxylamine/Acetate Solutions (B)
Table 4. Effect of Hydroxylamine/Acetate Solution on Stage 2b Reaction Mixtures (HPLC)
HPLC Analysis of
Stage 2a RM (area%)
HPLC Analysis of
pH
Stage 2b RM (area%)2
Entry
Range1
18
9
1
9
18
22.4
ND
ND
ND
20
96.9
95.7
96.5
94.5
2.3
2.1
2.0
3.9
3.2ꢀ5.4
5.5ꢀ5.8
6.1ꢀ6.5
5.5ꢀ6.1
69.7
92.3
91.6
89.9
4.91
2.78
2.63
4.67
2.96
0.62
0.11
0.50
1
2
3
4
1
2
RM, Reaction Mixture. pH range throughout addition of 18/NMP to buffered NH2OH·HCl solution.
HPLC
analysis performed prior to water addition. ND, Not Detected.
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Table 5. Physical Characterization of 1 Form A and 1 Form B
5
6
7
8
Moisture Sorption Analysis§
Microscopy
Onset
Decomposition
Temperature*
XRPD
Pattern
PostꢀDVS
XRPD
DSC (°C)
60% RH 90% RHǂ
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
60
Birefringent,
roundꢀ and
needleꢀshaped
particles.
Particle size:
20ꢀ100 ꢁm.
Birefringent,
needleꢀshaped
particles.
114.2 °C
(endotherm);
183.7 °C
1
0.06 wt% 0.004 wt%
Form A
185.5 °C
Form A
(exotherm)
109.2 °C
(endotherm);
184.4 °C
1
0.3 wt%
0.2 wt%
Form B
166.2 °C
Form B
Particle size:
10ꢀ50 ꢁm.
(exotherm)
XRPD, Xꢀray powder diffraction; DSC, differential scanning calorimetry; RH, relative humidity. * Determined by
thermogravimetric analysis (TGA). § Determined by dynamic vapor sorption (DVS). ǂ The slightly lower wt%
moisture measured at 90% RH is likely related to system error of the internal balance.
Table 6. The Effect of Coꢀsolvent on Filtration Rate of Crude 1
HPLC
Volume,
Preꢀ/Postꢀ
(mL)2
Filter/Wash
Time
Analysis
(area%)
Coꢀ
Solvent
Water
(mL)
Aging
Time
Recovery
(%)
Entry1
(min)3
1
20
3.2
1.9
4.3
0.5
0.9
2.9
2.6
3.3
1.7
IPAc
IPAc
0/40
10/30
10/30
10/30
40/0
120/0
120/0
120/0
120/0
200
200
200
200
200
610
610
610
4505
16 h
16 h
16
30
96.8
98.2
95.7
99.5
99.1
97.1
97.4
96.7
98.1
90
94
90
88
957
94
96
94
90
1
2
3
4
5
6
7
8
9
MTBE
toluene
toluene
toluene
toluene
toluene
toluene
16 h
35
16 h
5
20 min
2 min
6 min
1 h
5
204
35
35
2 h
<1 min6
1 Entries 1ꢀ6 were performed on 10 gꢀscale; entries 7ꢀ9 were performed on 30 gꢀscale. 2 Listed as preꢀ/postꢀaddition
of water. The suspensions were aged for the listed time at ambient temperature prior to filtration. After filtration,
3
4
the solids were washed with water (3 × 50 mL) the MTBE (2 × 50 mL). Prior to MTBE washing, wet cake
dimensions were 4.0 cm (height, H) × 9.5 cm (diameter, D); postꢀwashing dimensions: 1.5 cm H × 9.5 cm D. 5 Preꢀ
cooled 12% brine. 6 Filter time excludes washes. 7 1 Form B.
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Table 7. Stage 3 Recrystallization of Crude 11
5
6
7
8
Filtrate Purity
(area%)
Solid Purity (area%)
MP 19 20 MP
Citric
Acid2
Time Recovery
Entry
Water3
(h)
(%)
1
1
19
20
9
0.1
0.1
0.1
0.1
0
1
5
5
2
2
2
6
78
80
64
68
ND 98.4 1.0 0.5 4.2 61.0 12.7 22.0
ND 99.1 0.9 ND 2.9 64.5 13.0 19.6
ND 100 ND ND 2.1 61.8 20.4 14.6
ND 100 ND ND 3.1 62.0 15.5 18.7
1
2
3
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
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47
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51
52
53
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57
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60
1 Crude 1 purity: 96.7 area% 1, 2.2 area% 19, 1.1 area% 20. 2 wt/v% ACN. 3 v/v% ACN. ND, Not Detected.
Table 8. Temperature Cycles Applied for the Recrystallization of Crude 1.
Cycle 1
Cycle 2
Cycle 3
Cycle 4
Temp
(°C)
23
45
45
40
40
20
20
Temp
(°C)
20
Temp
(°C)
20
Temp
(°C)
20
45
45
35
35
10
na
Time
(h)
Time
Time
(h)
Time
(h)
(h)
0
1
0
1
2
2.5
3.5
7.5
11.5
0
1
0
45
45
1
1.5
2.5
3.5
7.5
11.5
45
30
30
20
1.5
2.5
3.0
9.0
na
45
30
40
20
1.5
2
3
8
na
20
na
na, not applicable.
Scheme 5. Final Synthetic Process for the Manufacture of 1
O
O
Stage 1a:
MsCl, DIPEA
O
CO2H
CO2H
HO2C
O
HO2C
THF, 15-20 °C
O
O
n
Suberic Acid (11)
Suberic Acid Poly-anhydride Mixture
O
O
O
Stage 1b:
Methyl Paraben, DIPEA
Stage 2a:
IBCF, NMM
O
O
OH
O
O
O
O
THF, 15-20 °C
NMP, -10 °C
MeO2C
MeO2C
(39%)
9
18
Stage 2b:
Stage 3:
4% water/ACN
0.1% citric acid
O
NH2OH.HCl, HOAc,
NaOAc, NMP, -10 °C
OH
O
N
H
SHP-141 (1)
O
45 °C then 10 °C
(76%)
(75%)
MeO2C
Crude 1
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