Development of a Multi Kilogram-Scale, Tandem Cyclopropanation Ring-Expansion Reaction en Route to Hedgehog Antagonist IPI-926

Article abstract of DOI:10.1021/acs.oprd.6b00048

The formation of the d-homocyclopamine ring system in IPI-926 is the key step in its semisynthesis and proceeds via a chemoselective cyclopropanation followed by a stereoselective acid-catalyzed carbocation rearrangement. In order to perform large-scale cyclopropanation reactions, we developed new iodomethylzinc bis(aryl)phosphate reagents that were found to be both effective and safe. These soluble reagents can be prepared under mild conditions and are stable during the course of the reaction. Importantly, they have favorable energetics relative to other cyclopropanating agents such as EtZnCH₂I. Herein, we describe the process optimization studies that led to successful large-scale production of the d-homocyclopamine core necessary for IPI-926.

Full text of DOI:10.1021/acs.oprd.6b00048

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Full Paper
Development of a Multi Kilogram-Scale, Tandem Cyclopropanation
Ring-Expansion Reaction En Route to Hedgehog Antagonist IPI-926
Brian C. Austad, Andrew Hague, Priscilla White, Stephane Peluso, Somarajan Nair,
Kristopher Depew, Michael Grogan, André B. Charette, Lin-Chen Yu, Caroline D. Lory,
Louis Grenier, André Lescarbeau, Benjamin S. Lane, Richard Lombardy, Mark Behnke,
Nii Koney, James R. Porter, Matthew J. Campbell, Jeanne Shaffer, Jimin Xiong, Joseph
C. Helble, Michael A Foley, Julian Adams, Alfredo Castro, and Martin R. Tremblay
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00048 • Publication Date (Web): 29 Mar 2016
Downloaded from http://pubs.acs.org on March 29, 2016
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Development of a Multi KilogramꢀScale, Tandem
Cyclopropanation RingꢀExpansion Reaction En
Route to Hedgehog Antagonist IPIꢀ926.
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Brian C. Austad,1 Andrew B. Hague, Priscilla White, Stéphane Peluso, Somarajan J. Nair,
Kristopher M. Depew, Michael J. Grogan,2 André B. Charette,3 Lin-Chen Yu, Caroline D. Lory,
Louis Grenier, André Lescarbeau, Benjamin S. Lane, 4 Richard Lombardy,5 Mark L. Behnke,6 Nii
Koney,7 James R. Porter, Matthew J. Campbell, Jeanne Shaffer, Jimin Xiong, Joseph C. Helble,
Michael A. Foley,8 Julian Adams, Alfredo C. Castro, and Martin R. Tremblay*
Infinity Pharmaceuticals, Inc. 784 Memorial Drive, Cambridge, Massachusetts 02139, United
States
1
Present address: Karyopharm Therapeutics, 85 Wells Ave., 2nd floor, Newton, MA 02459
2 Present address: Eli Lilly and Company, Indianapolis, IN 46285
3
Department of Chemistry, University of Montreal, Roger Gaudry Building, Dꢀ644, Faculty of Arts and Sciences,
2900 Edouard Montpetit Blvd. Montreal, P.O. Box 6128, Station Downtown QC H3C 3J7
4 Present address: Agios, 88 Sidney Street, Cambridge, MA 02139
5 Johnson Matthey Pharma Services, 25 Patton Rd, Devens, MA 01434
6 Present address: National Center for Advancing Translational Sciences, 31 Center Drive, Suite 3B11
Bethesda, MD 20892
7 Present address: Mount Sinai St. Luke's Hospital, 1111 Amsterdam Avenue, New York, NY 10069
8
Present address: TriꢀInstitutional Therapeutics Discovery Institute, Inc. Belfer Research Building, Weill Cornell
College, 413 E 69th St, New York, NY 10021
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Keyword: Cyclopropanation; SimmonsꢀSmith; cyclopamine; cycloheptane; cycloheptene; ring
expansion; carbenoid; homocyclopamine; carbocation rearrangement; iodomethylzinc phosphate
saridegib; IPIꢀ926
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ABSTRACT
The formation of the Dꢀhomocyclopamine ring system in IPIꢀ926 is the key step in its semiꢀ
synthesis and proceeds via a chemoselective cyclopropanation followed by a stereoselective
acidꢀcatalyzed carbocation rearrangement. In order to perform large scale cyclopropanation
reactions, we developed new iodomethylzinc bis(aryl)phosphate reagents that were found to be
both effective and safe. These soluble reagents can be prepared under mild conditions and are
stable during the course of the reaction. Importantly, they have favorable energetics relative to
other cyclopropanating agents such as EtZnCH2I. Herein, we describe the process optimization
studies that led to successful large scale production of the Dꢀhomocyclopamine core necessary
for IPIꢀ926.
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INTRODUCTION
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Malignant activation of the Hedgehog pathway is implicated in multiple cancer settings.1
Cyclopamine (1), a plant steroidal Veratrum alkaloid, is an antagonist of the Hedgehog pathway
and has been used as a biological tool to interrogate the pathway.2 However, cyclopamine’s
modest inhibitory activity on the pathway and poor pharmaceutical properties make it subpar as a
therapeutic agent. A few years ago, our group reported Dꢀhomo analogs of cyclopamine with
improved stability, potency and/or solubility.3 This work led to the identification of a novel,
semiꢀsynthetic analog of cyclopamine (IPIꢀ926, 2) with potent activity on the Hedgehog
pathway, significant antiꢀtumor activity, and attractive pharmaceutical properties.4 IPIꢀ926 was
evaluated in phase I and II clinical trials.
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The clinical development of IPIꢀ926 required the manufacture of multiꢀkilogram quantities of
drug substance. The formation of the Dꢀhomocyclopamine ring system in IPIꢀ926 is the crux in
its semiꢀsynthesis and proceeds via a chemoselective cyclopropanation followed by
stereoselective acidꢀcatalyzed rearrangement. In order to perform large scale cyclopropanation
reactions, we developed new iodomethylzinc bis(aryl)phosphate reagents that were found to be
both very effective and safe. Herein, we describe the process optimization studies that led to
successful large scale production of the Dꢀhomocyclopamine core necessary for IPIꢀ926.
METHODS AND RESULTS
The overall synthetic scheme for the production of IPIꢀ926 is captured in Scheme 1.5
Cyclopamine (1) is first converted to either bisꢀprotected cyclopamine analog 3a or 3b, which
can be used as substrates for the cyclopropanation ringꢀexpansion chemistry to give the sevenꢀ
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membered Dꢀring compounds 4a or 4b, respectively. After evaluation of various protecting
groups, we found Nꢀcarbamates and Oꢀesters/carbonates 3a and 3b to be superior for a number
of reasons, including ease of synthesis/removal, crystallinity/stability of intermediates, and
compatibility with Znꢀcarbenoid species. While 3a was a substrate of choice during the early
optimization phase of the cyclopropanation reaction, bisꢀCbz substrate 3b proved to be the
preferred intermediate due to streamlined downstream processing to Dꢀhomocyclopamine 5.
Oppenauer oxidation of 5 into 6 sets the stage for stereoselective reduction of the enone,6
methanesulfonamide installation,7 and finally salting of the piperidine ring8 to produce the active
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pharmaceutical ingredient 2.
This paper focuses primarily on the Dꢀring homologation
chemistry whereas the optimization of the largeꢀscale production of 2 from 6 will be the topics of
other publications.
Early Optimization
The gramꢀscale synthesis of 6, involving a twoꢀstep SimmonsꢀSmith cyclopropanation and
Lewis acidꢀmediated ring expansion, was reported by our discovery group.9 At the time, the key
SimmonsꢀSmith cyclopropanation step suffered from multiple issues that hampered scalability:
1) requirement for large excess of Zn carbenoid (~10 eq) mostly due to decomposition of the
reagents and subsequent workꢀup challenges; 2) decomposition of the starting material; 3)
heterogeneous reaction mixture; and 4) moderate overall yield. Most importantly, there were
considerable safety concerns with these types of Zn carbenoid species10 with reports of
explosions. We explored alternative cyclopropanating reagents such as Al carbenoids,11
transition metal catalyzed diazomethane decomposition,12 as well as haloformꢀderived
carbenes.13 However, none of these alternatives gave the desired transformation in any practical
yield on our substrates. Consequently, various Zn carbenoids were evaluated, particularly
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Furukawa’s conditions (Et2Zn, CH2I2),14 Shi’s conditions15 (Et2Zn, CH2I2, TFA), Merck’s
modifications of Shi’s protocol (Et2Zn, CH2I2, TCA),16 and Charette’s iodomethylzinc
phosphate reagents.17 All of these conditions converted substrate 3a into 4a relatively cleanly,
but required >5 equivalents of reagent to obtain useful conversions. Substrates 3a and 3b proved
to be quite sensitive to Lewis acidic conditions due to the cleavage of the spirotetrahydrofuran Cꢀ
O bond and subsequent aromatization of the Dꢀring (Scheme 2).18 Consequently, a delicate
balance between substrate stability and reactivity of the cyclopropanating agent needed to be
achieved. At the onset, two attractive features of the iodomethylzinc phosphate reagents sparked
our interest in pursuing further optimizations: 1) relative stability of the reagent since
iodomethylzinc phosphate could potentially be isolated as solid;17 2) ability to tune the reactivity
and physicochemical properties of the Zn carbenoid by modifying the phosphate ligand. The
major drawback of the known iodomethylzinc diphenylphosphate reagent was the poor solubility
of the carbenoid in solvents which dissolve 3a or 3b, resulting in a heterogeneous mixtures. We
pursued an optimization effort aiming to develop a volume efficient, homogeneous
cyclopropanation conditions by modification of the phosphate portion of the reagent. A number
of bis(aryl) and bis(alkyl) phosphoric acids were thus tested in the cyclopropanation reaction
using 3a as substrate (Table 1). Generally, iodomethylzinc bis(aryl)phosphates had better
reactivity toward our substrate than iodomethylzinc bis(alkyl)phosphates. The nature of the
substitution on the phenyl was explored to modulate the physicochemical properties such as a)
solubility of both the phosphate and the resulting carbenoid in dichloromethane, b) the ease of
removal after the reaction through basic aqueous washes, and c) crystalline properties of the
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phosphate raw material.
Gratifyingly, all these criteria were met by bis(2,6ꢀ
dimethylphenyl)phosphate (Table 1, entry 2), which was selected for further investigations.
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Although not commercially available, bis(2,6ꢀdimethylphenyl)phosphoric acid can be readily
prepared on large scale from reaction of 2,6ꢀdimethylphenol with phosphoryl chloride and
recrystallized to high purity.
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Both substrates 3a and 3b performed very well under these representative cyclopropanating
conditions (see Experimental section for all details): neat Et2Zn (3.05 eq) is charged to a cooled
solution of DCM (5 volumes, below ꢀ10 °C vide infra), then the mixture is cooled to ꢀ20 °C
before addition of a solution of bis(2,6ꢀdimethylphenyl)phosphoric acid (3.1 eq) in DCM (11.5
volumes). The solution is then warmed to 0 °C before the addition of substrates 3a or 3b (1 eq)
in DCM (3 volumes). Finally, CH2I2 (3.1 eq) in DCM (0.2 volume) is charged to initiate the
formation of the carbenoid and cyclopropanation reaction. The homogenous mixture is stirred at
30 °C and typically proceeds in >90% conversion after 5 h.
Calorimetry and safety assessment
With optimized cyclopropanating conditions for substrates 3a and 3b in hand, we first
measured the energetics of the various components of the reaction using differential scanning
calorimetry (DSC). A solution of Et2Zn in anhydrous DCM recorded an onset temperature of
decomposition at 83 °C by DSC (ramp rate of 2 °C/min) releasing energy of 222 J/g of total
mass. Solutions of Et2Zn/DCM/bis(2,6ꢀdimethylphenyl)phosphoric acid and Et2Zn/DCM/
bis(2,6ꢀdimethylphenyl)ꢀphosphoric acid/CH2I2 recorded no significant decomposition event up
to 160 °C and 120 °C, respectively. Based on these results, it was determined that the preparation
of Et2Zn/DCM solution could be safely operated at ꢀ10 °C which provides a safety window of 90
°C, whereas the cyclopropanating agent can be handled safely at room temperature.
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We then measured the heat generation of the reaction performed under Furukawa’s conditions
compared with the optimized conditions using SuperCRC calorimeter (Omnical). For
Furukawa’s conditions, the order of addition was Et2Zn, 3b, and then CH2I2. As expected, the
addition of CH2I2 to Et2Zn generated a substantial exotherm ranging from 734 – 870 kJ/mol of of
3b (N=2) (Figure 2A). Under adiabatic conditions, this exotherm would translate to an increase
in the internal temperature averaging 85 °C. For the optimized conditions, the order of addition
was Et2Zn, bis(2,6ꢀdimethylphenyl)phosphoric acid, 3b, and then CH2I2. The addition of
phosphate to diethylzinc generated an exotherm ranging from 489 – 537 kJ/mol of 3b (N=2),
whereas an exotherm (224 – 360 kJ/mol of 3b, N=2) was observed during the addition of CH2I2
(Figure 2B). From a temperature control standpoint, these exotherms were deemed controllable
since they would correspond to adiabatic temperature rise of 25 °C and 11 °C, respectively. The
energy released during the generation of the optimized cyclopropanating reagent was not
insignificant, but is broken down into two separate events. The acid/base reaction between
diethylzinc and the phosphate was the most exothermic event, which could be controlled by the
rate of phosphate addition. Of note, nitrogen sweeps were used to prevent accumulation of
ethane, which was then carried for scrubbing, dilution, or burning. The energy associated with
the alkyl transfer between (ArO)2P(O)OZnEt and CH2I2 was significantly lower compared to
Et2Zn. The formation of EtZnCH2I and Zn(CH2I)2 is typically accompanied with significant
decomposition and side reactions, which could also pose a safety concern.10a Consequently, the
iodomethylzinc bis(2,6ꢀdimethylphenyl)phosphate carbenoids were deemed safer, less energetic,
alternatives to the Furukawa process and thus chosen for scaleꢀup.
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One-pot ring expansion protocol
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The initial gramꢀscale synthetic route involved a twoꢀstep procedure to convert 3a into 4a.5, 9
After cyclopropanation, the cyclopropyl analog of 3a was isolated and then treated with BF3ꢀ
OEt2 to achieve the ring expansion to the sevenꢀmembered Dꢀring analog. After careful analysis
of other products generated under various cyclopropanating conditions, it was determined that
ringꢀexpanded products were minor byꢀproducts under those conditions. This observation
pointed to the possibility of a oneꢀpot procedure in which either in situ generated ZnX2 Lewis
acids or organic acids could be used directly at the end of the cyclopropanation reaction to cause
the desired ring expansion. To this end, we found the addition of Zn(OTf)2, and various alkyl or
aryl sulfonic acids to readily effect the ring expansion of 4a or 4b in situ after the
cyclopropanation was complete. Ultimately, stoichiometric methanesulfonic acid (MsOH) was
chosen based on conversion, purity profile, and ease of removal. Importantly, the addition of
MsOH to the cyclopropanation reaction mixture generated only a modest exotherm (174 kJ/mol
of 3b). However, the reaction required cooling (ꢀ55 °C) during this addition to minimize the
formation of a cyclic carbamate impurity (8a and 8b, Scheme 3) when the Fꢀring nitrogen was
protected with the preferred Cbz protecting group.
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An isolation and purification procedure for 4a and 4b coming from the cyclopropanation ring
expansion reaction was developed. A morpholine quench was implemented to mop up alkylating
byꢀproducts (LꢀZnCH2I, CH3I, EtI, and HI) and avoid occupational exposure to harmful and
potentially genotoxic species. Zinc salts, the aryl phosphate, and MsOH were removed by
aqueous washes (2N HCl followed by 4.8 wt% Na2CO3, 4.8 wt% Na2SO3 and 4.8 wt% NaCl).
The reaction mixture was then concentrated and solvent exchanged for the crystallization of 4b
from MeOH/IPA.
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Kilogram scale cyclopropanation ring expansion: reproducibility and key parameters
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With an efficient and safe oneꢀpot procedure in hand, a number of kilogram scale batches were
run to assess the performance and reproducibility of the conditions (Figure 3, entries 1ꢀ6). From
1 kg to 10 kg, the cyclopropanation ringꢀexpansion procedure proved to be very reproducible in
terms of yield (70ꢀ80%) and purity (95ꢀ97%) of 4b. Unfortunately, as the scale increased, the
cyclopropanation step began to stall frequently (Figure 3, entries 7ꢀ14). Although the stalled
reaction could be salvaged by charging fresh cyclopropanating reagent, this operation increased
plant time and complexity of the fixed equipment set up considerably. Root cause analysis and
followꢀup experiments determined that an aging period after the phosphate addition was
necessary to promote a more robust reagent preparation and a faster cyclopropanation reaction.
Consequently, a twoꢀhour hold period (before adding 3b) was implemented for subsequent
campaigns (Figure 3, entries 12ꢀ19). In these eight campaigns, the cyclopropanation step did
not stall except for one instance when CH2I2 was undercharged due to a line priming issue. This
issue was addressed with the use of a stainless steel tank fitted onto a balance to ensure the
proper weight was delivered after the system had been primed and rinsed.
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A tight operating range of ± 0.5 weight % of Et2Zn, phosphate, and CH2I2 was preferred to
ensure only the desired iodomethylzinc bis(aryl)phosphates reagent was formed and for
consistent reactivity. This tight control was a challenge in metering of pyrophoric neat
diethylzinc, and the order of addition (Et2Zn, phosphate, 3b, then CH2I2) exacerbated the
problem since one has to carefully control the stoichiometry while the substrate is already
committed to the reaction. We thus tested an alternative order of addition (Et2Zn, phosphate,
CH2I2, then 3b) and found that the cyclopropanating reagent could be prepared ahead of time and
used up to 19 hours later or charged directly into the substrate. Importantly, the new addition
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order could withstand charge fluctuations of ± 5 wt% without adversely affecting the outcome of
the cyclopropanation reaction (Figure 4). Employing the new order of addition, the
cyclopropanation progressed with as little as 2.5 equivalents of active reagent whereas the
reaction did not reach completion when 2.0 equivalents were used (Figure 5A). Interestingly, the
rate of cyclopropanation under the new order of addition was significantly better than the old
order even when suboptimal conditions (2.0 equivalents) were used (Figure 5B). Calorimetry
measurements of the CH2I2 addition to the (ArO)2P(O)OZnEt in the absence of substrate 3b
demonstrated a comparable exotherm to earlier work in the presence of substrate.19 The
subsequent addition of 3b in solution to the reaction mixture was minimally exothermic (0.37
kJ/mol of 3b). Based on the results of the reaction calorimetry, the reverse addition (generation
of the cyclopropanating reagent by addition of diiodomethane before the dosage of 3b) was
suitable to carry out the reaction in a safe manner. As a result, this process change was
implemented and performed on 33 kg scale (Figure 3, entries 20ꢀ26). Overall, this last change
improved the robustness of the oneꢀpot cyclopropanation ring expansion process and offered
greater flexibility in the plant.
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CONCLUSION
In conclusion, a robust and scalable oneꢀpot cyclopropanation ring expansion reaction
applicable to the Dꢀring homologation of cyclopamine has been developed. We found that
iodomethylzinc bis(aryl)phosphate carbenoids could be used safely and efficiently on multiꢀ
kilogram scale. Aside from typical key parameters such as oxygen/moisture sensitivity, quality
of starting materials, and physical properties of the phosphate, achieving the right stoichiometry
and order of addition of reagents were particularly important. Once the cyclopropanating agent
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is formed with the right ratio of reagents, the stability is excellent and can support smooth
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conversion of structurally complex and very acid labile allylic ether 3b.
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The robustness of this reaction and the stability of the iodomethyl bis(2,6ꢀ
dimethylphenyl)phosphate carbenoid allow for its use in radiosynthesis. To demonstrate this,
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substrate 3a was successfully converted to Cꢀ4a in acceptable yield and good purity using the
cyclopropanation/ring expansion conditions described above (Scheme 4). Isotopically 14Cꢀ
labeled diiodomethane has been used for Eschenmoser salt formation20 and dialkylation,21 while
3
SimmonsꢀSmith cyclopropanation reactions have been reported using 13Cꢀlabeled22 and Hꢀ
14
diiodomethane.23 To our knowledge, a cyclopropanation reaction using CꢀCH2I2 has not been
previously reported.
Although we have not looked at extending the scope of this reaction beyond cyclopamine
substrates, these reagents have attractive properties (soluble, stable, safe, and easy to remove)
and expand the pharmaceutical chemists’ toolbox.
Experimental section
General methods. Commercial reagents and solvents were used as received without further
purification or drying. All experiments involving waterꢀsensitive compounds were carried out
under argon and scrupulously dry conditions, using commercially available anhydrous solvents.
Thinꢀlayer chromatography was performed on glassꢀbacked precoated Merck silica gel (60 F254)
plates, and compounds were visualized using UV light, ceric ammonium molybdate or 2%
aqueous potassium permanganate solution. Silica gel column chromatography was carried out
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using Merck silica gel 60. Flash chromatography was run using silica gel (200ꢀ400 mesh) from
Sorbent Technologies. The purity of tested compounds was determined by analytical liquid
chromatography (HPLC) performed a number of different methods captured in the supporting
information. 1HꢀNMR spectra were recorded on a Bruker 400 spectrometer (400 MHz).
Chemical shifts are reported in ppm with the solvent resonance as the internal standard (CHCl3: δ
7.26). Data are reported as follows: chemical shift, integration, multiplicity (s=singlet,
d=doublet, dd=doublet of doublet, ddd=doublet of doublet of doublet, t=triplet, q=quartet,
br=broad, m=multiplet), and coupling constants (Hz). 13CꢀNMR spectra were recorded on a
Bruker 400 spectrometer (100 MHz) with complete proton decoupling. Chemical shifts are
reported in ppm with the solvent as the internal reference (CDCl3: δ 77.16). Electroꢀspray
positive ionization mass spectrometry was performed on samples using a reversed phase HPLCꢀ
MS system. The system was equipped with a Diode Array UV detector, a single quadrupole
mass spectrometer, and utilized the above mentioned reversedꢀphase HPLC gradient method.
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Cyclopamine extraction and isolation: Cyclopamine was obtained from Veratrum californicum
harvested in August. The samples were airꢀdried and milled to fine particles and extracted
according to A) methods adapted from Keelers and coꢀworkers,24 B) supercritical fluid extraction
,25 or C) liquidꢀliquid extraction.26 Cyclopamine can be recrystallized to purity in hot MeOH (30ꢀ
40 volumes). This protocol typically delivers 1ꢀ4 g of cyclopamine per kg of dry biomass.
Typical RPꢀHPLC (Method A) purity of cyclopamine obtained in this process exceeds 95 a/a%.
Synthesis of Cbz-Obt (3-Cbz-benzotriazole-1-oxide): 1ꢀHydroxybenzotriazole hydrate (70.9
kg, 463.4 mol, 1 eq.), dichloromethane (876 kg), and triethylamine (55.6 kg, 549.5 mol, 1.2 eq)
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are added to a reactor. The content of the reactor is cooled to 0 to 5 °C. Benzyl chloroformate
(87.0 kg, 510 mol, 1.1 eq) is added. The reaction solution is stirred for 4 hours, after which, it is
sampled for thin layer chromatography. Upon the completion of the reaction, water (192 kg) and
dichloromethane (600 kg) are added and the reaction is warmed to 15 °C. The quenched reaction
is settled and the aqueous layer is removed. The organic layer is washed twice with water (192
kg for each wash) to remove any triethylamine hydrochloride. The washed organic layer is
concentrated to a slurry and solvent exchanged to ethyl acetate until the dichloromethane content
in the distillate is below 5%. The material is then concentrated to a thick slurry and filtered. The
product collected is washed with ethyl acetate and dried under vacuum. 100.9 kg of 3ꢀCbzꢀ
benzotriaoleꢀ1ꢀoxide is produced at 81% yield. Mp = 107 °C; 1H NMR (400 MHz, Chloroformꢀ
d) δ 8.20 (d, J = 8.4 Hz, 1H), 8.00 (dt, J = 8.4, 1.0 Hz, 1H), 7.75 (ddd, J = 8.4, 7.2, 1.1 Hz, 1H),
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7.58 – 7.47 (m, 3H), 7.47 – 7.37 (m, 3H), 5.55 (s, 2H); C NMR (101 MHz, CDCl3) δ 147.42,
133.86, 133.52, 132.87, 132.82, 129.35, 129.00, 128.91, 126.45, 115.89, 115.23, 70.70; LRMS:
m/z = 270.4 [M + H]+; HPLC purity (method B): 99.9 a/a%.
Synthesis of Bis(2,6-dimethylphenyl)phosphoric acid: To a 500ꢀgallon reactor, pyridine
(620 kg) and 2,6ꢀdimethylphenol (256 kg, 2096 mol, 1.98 eq) are charged. Phosphoryl chloride
(162 kg, 1057 mol, 1 eq) is added over 2ꢀ3 hours in order to maintain the internal temperature
between 65 and 70 °C. The resulting suspension is stirred for 1ꢀ2 hours at the same temperature
and then cooled to 8ꢀ12 °C. The reaction is sampled for inꢀprocessꢀcontrol by HPLC (acceptance
criterion: 2,6ꢀdimethylphenol < 5%). Upon the reaction completion, the reaction mixture is
filtered and the filter cake is washed with 23.5 kg of pyridine. The combined filtrate is stirred at
14ꢀ18 °C. Water (1249 L) is added over 1ꢀ2 hours and the resulting suspension is warmed to 20ꢀ
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25 °C. Dichloromethane (420 kg) is then added. The mixture is settled and the phases are
separated. The aqueous layer is washed twice with dichloromethane (550 kg for each wash). The
combined organic layers are washed with aqueous HCl solution (1059 kg conc. HCl in 984 L of
water) three times and water (519 L) once. Water (757 L) is then added to the organic layer,
followed by 227 L of 20% aqueous NaOH solution to adjust the pH to 13ꢀ13.5. The mixture is
stirred for about an hour and the phases are separated. The aqueous layer is acidified to pH 0.5ꢀ
1.0 by the addition of 130ꢀ140 kg of 37% HCl solution. The resulting offꢀwhite suspension is
stirred for 90 min. The supernatant is decanted and water (719 L) is added and stirred well for
about an hour. The decanting, water addition, and stirring are repeated. After the final decanting
of supernatant, wet white solid is left in the reactor for recrystallization. First Recrystallization:
2ꢀPropanol (790.5 kg) is charged to the wet white solid in the reactor. Water (1500 L) is added
and the whole mixture is transferred to a 2000ꢀgallon reactor. The mixture is heated at 78ꢀ82 °C
for 70 minutes to give a hazy solution. The solution is cooled to 8ꢀ12 °C in about 6 hours and
stirred for 3ꢀ5 hours. The suspension is filtered in a centrifuge filter and the filter cake is washed
with a 2:3 mixture of 2ꢀpropanol and water (382 L in two portions). The wet product is dried
further to afford 245 kg of offꢀwhite solid of 99.96% purity by HPLC. Second recrystallization:
To the product from the last recrystallization, 2018 kg of cyclohexane is added. The mixture is
heated to 78ꢀ82 °C and a slightly hazy solution is formed. The solution is hotꢀfiltered through a
pressure filter with dicalite dish pack and heated with stream. The filtrate is cooled to 8ꢀ12 °C in
about 6 hours. The resulting white slurry is held at the same temperature for at least 40 hours.
The material is filtered in a centrifuge filter. The filter cake is washed with 150 kg of cold
cyclohexane and then spun for 50ꢀ70 minutes. The wet product is dried further at 40ꢀ50 °C under
vacuum for at least 24 hours. Crystallized bis(2,6ꢀdimethylphenyl)phosphoric acid (230 kg) is
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obtained at 72% yield. Mp = 143 °C; 1H NMR (400 MHz, Chloroformꢀd) δ 9.88 (s, 1H), 6.97 (s,
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6H), 2.18 (d, J = 1.1 Hz, 12H); C NMR (101 MHz, CDCl3) δ 148.25, 148.17, 130.52, 129.10,
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129.08, 125.37, 125.35, 16.91; P NMR (162 MHz, CDCl3) δ ꢀ7.87; LRMS: m/z = 307.5 [M +
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H]+; Purity method C: >99.9 a/a%
Compound 3a: Recrystallized cyclopamine 1 (14.1 g, 34.0 mmol, 1 eq) is dissolved in
anhydrous DCM (70 mL) and anhydrous MeOH (29 mL). The clear solution is cooled, and
triethylamine (10.4 g, 102.7 mmol, 3 eq) followed by benzyl chloroformate (6.20 g, 36.3 mmol,
1.1 eq) is added. After the addition is complete, the solution is stirred in the ice bath for 30 min.
Three portions of benzyl chloroformate (3x0.35 g, 3.46 mmol, 0.03 eq) are added over the 3 h.
The reaction is slowly quenched with water (71 mL), while maintaining the temperature below
20 °C. The mixture is stirred for 15 min before the layers are settled and separated. The organic
layer is dried over sodium sulfate and filtered. The combined filtrate is buffered with anhydrous
pyridine (30 mL), concentrated, and solvent exchanged with additional anhydrous pyridine (43
mL) and concentrated. The solution of the compound in pyridine (43 mL) is further diluted with
additional anhydrous pyridine (85 mL). Trimethylacetyl chloride (8.3 g, 68.7 mmol, 2 eq) is
added slowly to the reaction mixture, and the reaction is heated to 45 °C. The reaction is stirred
at 45 °C for 30 min. The reaction is cooled and quenched by the addition of anhydrous MeOH
(4.5 mL). The quenched reaction mixture is stirred at 23 °C for 40 min and then diluted with
toluene (97 mL) and is treated sequentially with water (35 mL) and a 10 wt% aqueous sodium
carbonate solution (100 mL). After vigorous stirring, the layers are separated and the organic
layer is washed twice with water (2x 100 mL), dried over sodium sulfate, and filtered. The filter
cake is rinsed with toluene (49 mL) and discarded. The combined filtrates are concentrated, and
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solvent exchanged with concentration to toluene (145 mL) and further concentrating to dryness.
The product is recrystallized from toluene and heptane. The crystalline product is isolated by
suction filtration, washed with cold heptane and dried to a constant weight to afford 15.1 g of 3a.
1H NMR (400 MHz, Pyridineꢀd5) δ 7.61 – 7.54 (m, 2H), 7.47 – 7.37 (m, 2H), 7.39 – 7.30 (m,
1H), 5.41 (s, 2H), 5.41 – 5.34 (m, 1H), 4.84 (ddd, J = 11.3, 11.1, 5.6 Hz, 1H), 3.79 (dd, J = 12.9,
4.2 Hz, 1H), 3.71 – 3.58 (m, 1H), 3.40 (dd, J = 10.3, 7.2 Hz, 1H), 3.15 (p, J = 7.2, 7.2, 7.0, 7.0
Hz, 1H), 2.96 (dd, J = 12.9, 8.3 Hz, 1H), 2.54 (ddd, J = 13.3, 4.9, 2.1 Hz, 1H), 2.40 (br t, J = 12.5
Hz, 1H), 2.26 – 2.02 (m, 4H), 2.01 –1.86 (m, 2H), 1.85 – 1.79 (m, 3H), 1.82 – 1.60 (m, 6H), 1.59
– 1.45 (m, 2H), 1.39 (ddd, J = 11.2, 10.8, 8.7 Hz, 1H), 1.29 (d, J = 1.5 Hz, 9H), 1.30 – 1.16 (m,
2H), 1.17 – 1.04 (m, 4H), 0.88 (d, J = 7.2 Hz, 3H), 0.87 (s, 3H); 13C NMR (101 MHz, Pyridineꢀ
d5) δ 178.02, 158.60, 142.84, 141.30, 138.08, 129.40 (2C), 128.93 (2C), 128.91, 127.88, 123.47,
85.10, 74.19, 73.50, 67.77, 64.43, 52.57, 51.98, 49.68, 42.64, 42.57, 39.20, 38.49, 38.38, 37.26,
36.13, 33.32, 31.74, 29.60, 29.20, 28.19, 27.74 (3C), 25.57, 20.65, 18.95, 14.35, 11.31; LRMS:
m/z = 630.7 [M + H]+
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Compound 3b: A suitable sized jacketed reactor, equipped with a temperature control unit,
distillation setꢀup, and a receiving vessel is flushed with nitrogen, then charged with
cyclopamine (11 kg, 1 wt, 1 eq), nꢀpropyl acetate (116.6 kg, 10.6 wt, 12 volumes), DMAP (1.63
kg, 0.148 wt, 0.5 eq) and triethylamine (8.12 kg, 0.738 wt or 1.016 volumes, 3 eq). The mixture
is stirred at 20 ± 5 °C, then CbzꢀObt (16.56 kg, 1.505 wt, 2.3 equiv.) is charged in three equal
portions (≈ 0.5 wt each) while maintaining the reaction at 20 ± 5 °C. After each charge, the
reaction is held for a minimum of 30 minutes. Once the charge is complete, the reaction is stirred
for a minimum of 3 hours at 20 ± 5 °C. Once the reaction is complete, the suspension is heated to
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50 ± 5 °C to obtain a homogeneous solution. Methanol (139.7 kg, 12.7 wt, 16 volumes) is slowly
added to the reaction while maintaining the reaction temperature at 50 ± 5 °C. The slurry is then
cooled to 0 ± 5 °C over 5 hours, then held at 0 ± 5 °C for ≥ 12 hours. The solids are collected by
vacuum filtration, followed by rinsing with methanol (49.5 kg, 4.5 wt, 5.7 volumes) and with
heptane (33 kg, 3.0 wt, 4.4 volumes). The solid is dried with warm nitrogen (40 °C) for ≥ 12
hours, then under vacuum at 40 °C for ≥ 8 hours. This procedure delivered 14.7ꢀ16.6 kg of 3b
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(N=10, 81.4%ꢀ91.8% yield). Mp = 152 °C; H NMR (400 MHz, Pyridineꢀd5) δ 7.56 (ddd, J =
15.7, 7.6, 1.5 Hz, 4H), 7.47 – 7.30 (m, 6H), 5.41 (s, 2H), 5.37 (s, 2H), 5.36 – 5.32 (m, 1H), 4.93
(d, J = 1.7 Hz, 2H), 4.71 (tt, J = 11.4, 4.7 Hz, 1H), 3.79 (dd, J = 13.0, 4.2 Hz, 1H), 3.63 (td, J =
10.9, 4.3 Hz, 1H), 3.39 (dd, J = 10.3, 7.2 Hz, 1H), 3.15 (t, J = 7.3 Hz, 1H), 2.95 (dd, J = 12.9, 8.2
Hz, 1H), 2.60 (ddd, J = 13.2, 5.0, 2.1 Hz, 1H), 2.48 – 2.37 (m, 1H), 2.24 – 1.92 (m, 6H), 1.82 (s,
3H), 1.80 – 1.45 (m, 7H), 1.36 (td, J = 11.2, 8.5 Hz, 1H), 1.28 – 1.04 (m, 5H), 0.88 (d, J = 6.8
13
Hz, 3H), 0.83 (s, 3H); C NMR (101 MHz, Pyridineꢀd5) δ 158.60, 155.47, 142.77, 140.88,
138.08, 136.93, 129.43, 129.40, 129.20, 129.10, 128.92, 127.91, 123.82, 85.08, 78.46, 73.49,
69.89, 67.77, 64.42, 52.50, 51.97, 49.68, 42.63, 42.50, 38.37, 38.29, 37.15, 36.12, 33.32, 31.70,
29.56, 29.19, 28.24, 25.54, 20.64, 18.83, 14.33, 11.30; LRMS: m/z = 680.7 [M + H]+; HPLC
purity (Method D) of 99.6ꢀ99.7 a/a %.
Compound 4a: Bis(2,6ꢀdimethylphenyl)phosphoric acid (10.65 g, 34.8 mmol, 3.1 eq) is dried
by concentration from anhydrous dichloromethane (42 mL) and held under a nitrogen
atmosphere. The phosphoric acid is then redissolved in anhydrous dichloromethane (110 mL). In
a separate flask, a solution of neat diethylzinc (4.17 g, 34.0 mmol, 3.0 eq) in anhydrous
dichloromethane (35 mL) is prepared and cooled to ꢀ25 °C. The phosphate solution is slowly
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transferred to the vessel containing the diethylzinc solution over 1 h, maintaining the temperature
at or below ꢀ10 °C. The clear ethylzinc phosphate solution is warmed to 0 °C and stirred for 15
min. Diiodomethane (9.25 g, 34.5 mmoles, 3.0 eq) is slowly added to the ethylzinc phosphate
solution, maintaining the reaction temperature between 0 and 5 °C. After the addition is
complete, the zinc carbenoid solution is stirred for an additional 20 min. In a separate flask,
compound 3a (7.20 g, 11.4 mmol, 1 eq) is dissolved in anhydrous dichloromethane (36 mL) and
transferred to the reaction flask. After the addition is complete, the ice bath is removed and the
reaction mixture is allowed to warm to rt. After 6 h the contents of the flask are cooled to ꢀ53 °C.
A solution of methanesulfonic acid (3.38 g, 35.2 mmol, 3.1 eq) in anhydrous dichloromethane (3
mL) is added, maintaining the reaction temperature below ꢀ45 °C. After 10 min, morpholine (20
g, 230 mmol, 20 eq) is added to the reaction mixture, maintaining the reaction temperature below
ꢀ40 °C. The reaction is allowed to warm to rt overnight. The morpholine salts are removed by
filtration and the filter cake rinsed with dichloromethane (22 mL). The combined filtrates are
washed with 2N aqueous hydrochloric acid (2x140 mL), 5% aqueous sodium bicarbonate (140
mL), 5% aqueous sodium bicarbonate (70 mL) and 5% aqueous sodium bisulfite (70 mL), and
brine (140 mL). The organic layer is dried over magnesium sulfate and filtered. Without going to
dryness, the dichloromethane solution is concentrated and solvent exchanged with methanol (280
mL). The suspension are chilled with an ice bath and stirred for 40 minutes. The solids are
isolated by filtration, washed twice with cold methanol (2x25 mL), and dried to a constant
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weight to afford 5.94 g of 4a. H NMR (400 MHz, Chloroformꢀd) δ 7.41 – 7.27 (m, 5H), 5.40
(dd, J = 4.8, 2.2 Hz, 1H), 5.20 – 5.07 (m, 2H), 4.58 (dddd, J = 11.3, 11.3, 4.8, 4.8 Hz, 1H), 3.82
(dd, J = 12.9, 4.3 Hz, 1H), 3.63 (ddd, J = 11.2, 10.1, 4.2 Hz, 1H), 3.09 (dd, J = 10.2, 6.2 Hz, 1H),
2.69 – 2.53 (m, 2H), 2.47 (d, J = 14.0 Hz, 1H), 2.42 – 2.32 (m, 1H), 2.33 – 2.08 (m, 5H), 2.06 –
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1.91 (m, 3H), 1.86 – 1.69 (m, 4H), 1.68 (s, 3H), 1.66 – 1.51 (m, 3H), 1.50 – 1.36 (m, 3H), 1.32 –
1.19 (m, 2H), 1.18 (s, 9H), 0.99 (s, 3H) 0.98 (d, J = 8.3 Hz, 3H), 0.91 (d, J = 7.0 Hz, 3H).
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Compound 4b: Equipment: Reaction Vessel A 2500 L (for diethylzinc solution and reaction)
is an appropriately sized, cryogenic capable jacketed reactor equipped with a temperature control
unit, a nitrogen/vacuum port, a charge port, an overhead stirrer and condenser. Reagent Vessel B
(for bis(2,6ꢀdimethylphenyl)phosphoric acid [BDMPP] solution) is an appropriately sized
jacketed reactor equipped with a temperature control unit, a nitrogen/ vacuum port, a charge port,
an overhead stirrer and condenser. Reagent Vessel C (for starting material compound 3b
solution) is an appropriately sized jacketed reactor equipped with a temperature control unit, a
nitrogen/ vacuum port, a charge port, an overhead stirrer and condenser. Reagent Vessel D (for
methanesulfonic acid solution) is an appropriately sized jacketed reactor equipped with a
temperature control unit, a nitrogen/ vacuum port, a charge port, an overhead stirrer and
condenser. Reaction set-up: Vessel B is charged with bis(2,6ꢀdimethylphenyl)phosphoric acid
(45.46 kg, 1.39 wt, 3.10 eq), then sealed and pressurized with nitrogen to 1.7 bar. The pressure
is released and a vacuum to 0.3 bar is pulled. This step is repeated twice, then the reactor is back
filled with nitrogen to 1 bar. Using residual vacuum and utilizing anhydrous techniques, charge
anhydrous dichloromethane (400 L, 13 volumes) to vessel B. The batch is agitated at 20 ± 5 °C
until all the solid has dissolved. Vessel B is sealed and pressurized with nitrogen to 1.7 bar. The
pressure is released to a minimum of 0.3 bar and held for ≥ 5 minutes, then nitrogen is back
filled to 1 bar. This step is repeated three more times. The diethylzinc cylinder is connected to
the transfer manifold and purged accordingly (see supporting information). The charge manifold
is connected to vessel A via a transfer line employing Swagelok fittings, and also to a cylinder of
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anhydrous dichloromethane, and a nitrogen source. A nitrogen sweep (2 – 5 psi) is applied
through the charge manifold, transfer line and reaction vessel A for a minimum of 12 hours.
Using residual vacuum and utilizing anhydrous techniques, anhydrous dichloromethane (500 L,
15 volumes) is charged to vessel A, which is then sealed and pressurized with nitrogen to 1.7 bar.
The pressure is released and a vacuum is pulled to 0.3 bar. This step is repeated twice, then
finished with nitrogen back fill to 1 bar. The batch is agitated under nitrogen, then cooled to ꢀ10
± 5 °C. Using low pressure nitrogen, diethylzinc (18.03 kg, 0.55 wt, 3.05 eq) charged into vessel
A. The manifold and transfer line are rinsed with anhydrous dichloromethane three times, the
rinses (30 L) directed into the reaction (vessel A). After each rinse the lines are flushed with
nitrogen for ≥ 2 minutes. The phosphate solution is slowly transfer from vessel B into the
reaction (vessel A), while maintaining the temperature ≤ 5 °C under nitrogen. This transfer
typically takes 4 hours and must not exceed 7 hours. Diiodomethane is charged (39.75 kg, 1.22
wt, 3.10 eq) to the reaction (vessel A), while maintaining the temperature ≤ ꢀ10 ± 5 °C. Vessel C
is charged with compound 3b (32.55 kg, 1 wt, 1.0 eq), then pressurized with nitrogen to 1.7 bar.
The pressure is released and a vacuum is pulled to 0.3 bar. This step is repeated twice, then
finished with a nitrogen back fill to 1 bar. Using residual vacuum and utilizing anhydrous
techniques, anhydrous dichloromethane is charged (65 L, 2 volumes) to vessel C and the material
is agitated at 20 ± 5 °C until it dissolves. The dichloromethane solution of compound 3b is
transferred from vessel C into the reaction (vessel A), while maintaining the temperature ≤ ꢀ10 ±
5 °C with stirring. Using residual vacuum and utilizing anhydrous techniques, vessel C is rinsed
with anhydrous dichloromethane (32 L, 1.33 wt, 1 volume). Once the transfer is complete, the
batch is warmed to 7.5 ± 2.5 °C and held with a nitrogen purge to the scrubber for 1 hour in
order to allow dissolved ethane to vent off. The batch is then warmed to 30 ± 5 °C while
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monitoring the internal pressure monitored every 5 °C. If the reactor pressure increases by 1 bar
above atmospheric pressure, suspend the warm up and vent the reactor with a nitrogen purge to
the scrubber over 1 hour. The batch is agitated at 30 ± 5 °C for ≥ 4 hours. After the reaction is
found to be complete, the batch is cooled to ꢀ 60 ± 5 °C. Ring-expansion: Using residual vacuum
methanesulfonic acid (13.9 kg, 0.424 wt, 3.0 eq) is charged to vessel D. Using residual vacuum
and utilizing anhydrous techniques, anhydrous dichloromethane (55.6 kg, 1.72 wt, 1.3 volumes)
is charged to vessel D. When the batch in vessel A reaches ≤ ꢀ 55 °C, the methanesulfonic acid
solution in vessel D is transferred to the batch (vessel A), while maintaining the temperature at ≤
ꢀ50 °C. Using residual vacuum and utilizing anhydrous techniques, anhydrous dichloromethane
is transferred (11.7 kg, 0.36 wt, 0.27 volumes) to vessel D, then the rinse is transferred to the
batch (vessel A). Quench: Once the ring expansion reaction is complete, morpholine (42.1 kg,
1.28 wt, 1.29 volumes, 10 eq) is charged to the batch (vessel A), while maintaining the
temperature at ≤ ꢀ 35 °C. The batch (vessel A) is then warmed to 20 °C and held for ≥ 1 hour.
The batch is washed with an aqueous solution of 2N hydrochloric acid (336 kg, 10.3 wt, 10
volumes, 13.6 eq) for ≥ 60 min at 20 – 25 °C. The agitation is stopped and the organic layer
(bottom layer) is separated from aqueous layer (top layer). The organic layer is transferred back
into the reactor (vessel A) and the washing step with 2N hydrochloric acid (336 kg, 10.3 wt, 10
volumes, 13.6 eq) is repeated for ≥ 15 min at 20 – 25 °C. The organic layer is washed with an
aqueous solution of 4.8 wt% sodium carbonate (338 kg, 10.5 wt, 10 volumes, 3.2 eq) for ≥ 15
min at 20 – 25 °C. The agitation is stopped, and the organic layer (bottom layer) is separated
from aqueous layer (top layer). The organic layer is transferred back into the reactor (vessel A).
The batch is washed with an aqueous solution of 4.8 wt% sodium sulfite (339.4 kg, 10.5 wt, 10
volumes, 2.7 eq) for ≥ 15 min at 20 – 25 °C. The agitation is stopped and the organic layer
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(bottom layer) is separated from aqueous layer (top layer). The organic layer is transferred back
into the reactor (vessel A). The batch is washed with purified water (326 kg, 10.5 wt, 10
volumes, 5.9 eq) for ≥ 15 min at 20 – 25 °C. The agitation is stopped, and the organic layer
(bottom layer) is separated from aqueous layer (top layer). The organic layer is polished filtered
and transferred into a 630 L reactor. The filtrate is distilled under vacuum to 2 ± 0.25 volumes,
while maintaining the batch temperature at ≤ 35 °C during the distillation. To the concentrated
filtrate is charged 2ꢀpropanol (97.7 kg, 3.0 wt, 3.82 volumes) and the batch is agitated at 20 ± 5
°C until the solution is homogeneous. Methanol (49.0 kg, 1.5 wt, 1.90 volumes) is then charged
into the batch over ≥ 10 minutes at 20 ± 5 °C. The mixture is agitated for 30 ± 5 minutes at 20 ±
5 °C to initiate nucleation. Then, more methanol (179.2 kg, 5.5 wt, 6.95 volumes) is charged
over 45 ± 15 minutes at 20 ± 5 °C. The batch is agitated at 20 ± 5 °C for ≥ 4 hours, then at 0 ± 5
°C for ≥ 4 hours. The solid is filtered off over a 30 micron nylon filter cloth and deꢀliquored with
nitrogen. The solid is washed with methanol (130.4 kg, 4 wt, 5.1 volumes), soaked for ≥ 10
minutes, then deꢀliquored with nitrogen. The solid is washed with heptane (130 kg, 4 wt, 5.8
volumes), soaked for ≥ 10 minutes, then deꢀliquored with nitrogen. The solid is dried with
nitrogen for ≥ 4 hours, then warm nitrogen (40 °C) for ≥ 12 hours while periodically stirring the
filter cake. The solid is dried under vacuum at 40 °C for ≥ 8 hours while periodically stirring the
filter cake. This procedure delivered 18.3ꢀ26.7 kg of 4b (N=7 on 33 kg scale, range 55ꢀ80%
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yield). Mp = 148 °C; H NMR (400 MHz, Pyridineꢀd5) δ 7.63 – 7.50 (m, 4H), 7.47 – 7.30 (m,
6H), 5.48 – 5.32 (m, 5H), 4.93 (s, 3H), 4.69 (tt, J = 11.3, 4.7 Hz, 1H), 3.95 (dd, J = 12.8, 4.3 Hz,
1H), 3.64 (td, J = 10.8, 4.1 Hz, 1H), 3.24 (dd, J = 10.1, 6.1 Hz, 1H), 2.88 (m, 1H), 2.70 – 2.56
(m, 2H), 2.52 (d, J = 14.1 Hz, 1H), 2.42 (td, J = 12.4, 2.9 Hz, 1H), 2.23 (dtd, J = 14.5, 6.7, 6.1,
3.2 Hz, 2H), 2.16 – 1.92 (m, 5H), 1.90 – 1.53 (m, 7H), 1.47 – 1.28 (m, 2H), 1.26 – 1.00 (m, 6H),
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0.90 – 0.77 (m, 6H); 13C NMR (101 MHz, Pyridineꢀd5) δ 158.20, 155.46, 141.67, 140.71,
138.10, 136.94, 129.43, 129.41, 129.20, 129.10, 129.02, 128.91, 124.58, 82.42, 78.53, 75.70,
69.87, 67.72, 63.06, 53.29, 52.75, 50.67, 50.46, 46.86, 44.37, 38.42, 38.38, 38.34, 38.14, 37.37,
32.42, 31.16, 29.37, 29.28, 28.24, 24.55, 19.81, 18.95, 11.18; LRMS: m/z = 694.7 [M + H]+;
HPLC purity (Method D) range of 95.6ꢀ97.2 a/a %.
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Compound 5 from 4a: In a round bottom flask, compound 4a (11.67 g, 18.1 mmol, 1 eq) and
20% palladium hydroxide on wet carbon (2.40 g, 1.71 mmol, 0.09 eq) are placed under a
nitrogen atmosphere and diluted with EtOAc (115 mL) and toluene (60 mL). The solution is
degassed with nitrogen (3x) with evacuation/purge cycles, and the process is repeated for
hydrogen. The suspension is vigorously stirred at 22 °C for 1.5 h. The hydrogen atmosphere is
replaced with nitrogen. Ethylenediamine (0.57 g, 9.5 mmol, 0.52 eq) is added to the reaction, and
the resulting mixture stirred for 20 min. The solution is filtered under nitrogen, and the filtrate is
washed with a 2% (wt/wt) aqueous solution of ethylenediamine (125 mL) then water (130 mL),
and then dried over sodium sulfate. The drying agent is removed by filtration and the filtrate is
concentrated to dryness under vacuum. The remaining solids are chased with toluene (2x55 mL)
on the rotary evaporator and the resulting material used without further purification in the next
step. The material from the previous step is dissolved in anhydrous dichloromethane (26 mL).
The resulting clear solution is added to a 1 M solution of DIBAL in dichloromethane (65 mL, 65
mmol, 3.6 eq) while maintaining the reaction temperature between ꢀ10 and ꢀ25 °C. After 30 min
the reaction is quenched with acetone (13 mL), maintaining the reaction temperature at or below
0 °C. After stirring the quenched reaction mixture for 20 min, it is added in portions to a flask
containing a cold, stirred solution of 20% (wt/wt) aqueous Rochelle salt (200 mL). The resulting
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gelatinous suspension is stirred at 23 °C for 15 h. After stirring, the clean layers are separated
and the aqueous layer back extracted with dichloromethane (30 mL). The combined organic
layers are washed with water (60 mL) and dried over sodium sulfate. The drying agent is
removed by filtration and discarded. The filtrate is concentrated under vacuum and solvent
exchanged to toluene (225 mL added in portions). The resulting solution is further concentrated
to a suspension (50 mL) and diluted with heptane (115 mL). The resulting mixture is heated until
turning homogeneous (92 °C). The solution is cooled slowly over 12 h to 15 °C, and then held
for an additional 16 h. The crystalline product is isolated by suction filtration, washed with
heptane (2x75 mL) and dried to a constant weight to afford 7.70 g of 5.
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Compound 5 from 4b: An sizeꢀappropriate reaction vessel, equipped with temperature
controller unit, a condenser, a nitrogen/vacuum port, a paddleꢀtype stirrer, and a hydrogen inlet,
is flushed with nitrogen then charged with 4b (25.3 kg, 1 wt, 1.0 eq) and 5% Pd/C (Johnson
Matthey type A503038ꢀ5, (9.3 kg, 0.368 wt, 0.06 eq of Pd metal) 50% wet catalyst). The vessel
is evacuated and back fill with nitrogen once. To the reaction vessel is charged tetrahydrofuran
(263 kg, 11.7 volumes or 10.4 wt; HPLC solvent grade inhibited w/ BHT) and deionized water
(32.9 kg, 1.3 volumes or 1.3 wt). The batch is stirred and the system is evacuated the system
until solvent is seen dripping from the condenser (pressure should be about ꢀ0.8 bar), and then
back fill with nitrogen. This process is repeated four times, then back fill with hydrogen. A
hydrogen pressure of 1 ± 0.1 bar should be used. The heterogeneous mixture is stirred rapidly at
20 ± 5 °C. Once the reaction is complete, the vessel is evacuated to ꢀ0.8 bar and refill with
nitrogen 3 times. To the reaction vessel is charged ethylenediamine (0.153 kg, 0.00605 wt, 0.07
eq) and Celite (9.3 kg, 0.368 wt). The suspension is stirred at 50 ± 2 °C for 15 min, then cooled
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to 20 ± 2 °C. The batch is filtered under nitrogen, collecting the filtrate in a vessel appropriate
for distillation. The Celite pad is rinsed with a preꢀmixed combination of tetrahydrofuran (51 kg,
1.9 vol or 1.69 wt) and deionized water (3 kg, 0.1 vol or 0.1 wt), then acetone (100 kg, 5 vol or
3.96 wt). The solvents were atmospherically distilled off using a 62 °C internal temperature set
point. Once the batch volume reaches ~5 volumes, additional acetone (539 kg, 19 vol or 15 wt) is
charged to the batch. This charge is made slowly to maintain the batch at ~5 volumes. Once the
acetone addition is complete, the distillation is continued until the internal temperature reaches
62 °C (jacket temperature set point 65 °C), or until the minimum stirable volume of 3ꢀ4 volumes
is reached. At this point, the internal temperature set point is changed to 50 °C, and heptane (138
kg, 8 vol or 5.5 wt) is simultaneously added in slowly. The batch stirred at 50 ± 5 °C for a
minimum of 1 hour, then cooled down to 20 ± 5 °C and held for a minimum of 2 hours. After the
hold time is complete, the batch is filtered and the product cake is washed with 5 volumes of a
preꢀmixed combination of acetone (25 kg, 1.25 vol, 0.99 wt) and heptanes (65 kg, 3.75 vol, 2.56
wt). The solid is dried with nitrogen for ≥ 1 hour, warm nitrogen (40 °C) for ≥ 12 hours, then
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under vacuum at 40 °C for ≥ 8 hours. Mp = 248 °C; H NMR (400 MHz, Pyridineꢀd5) δ 6.17 (s,
1H), 5.46 (dt, J = 4.2, 1.9 Hz, 1H), 4.93 (s, 1H), 3.93 – 3.80 (m, 1H), 3.51 – 3.40 (m, 1H), 3.12 –
3.03 (m, 1H), 2.82 – 2.67 (m, 2H), 2.66 – 2.49 (m, 2H), 2.40 – 2.25 (m, 3H), 2.19 (dt, J = 11.4,
3.8 Hz, 1H), 2.13 – 1.75 (m, 6H), 1.71 (m, 2H), 1.62 – 1.36 (m, 3H), 1.36 – 1.13 (m, 7H), 1.02
13
(d, J = 7.3 Hz, 3H), 0.93 (s, 3H), 0.85 (t, J = 6.9 Hz, 2H), 0.82 (d, J = 6.6 Hz, 3H); C NMR
(101 MHz, Pyridineꢀd5)) δ 143.14, 141.60, 124.80, 122.28, 82.93, 77.42, 71.84, 65.27, 55.79,
53.20, 51.02, 50.73, 45.86, 44.60, 43.50, 41.32, 39.35, 38.11, 37.58, 32.85, 32.60, 32.49, 32.11,
31.39, 29.67, 29.33, 24.70, 23.36, 19.56, 19.35, 14.71, 11.17; LRMS: m/z = 426.7 [M + H]+;
HPLC (Method A): 99.2 a/a%
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Compound 6: A size appropriate jacketed reactor is equipped with an overhead stirrer,
thermocouple, temperature control unit, a condenser, and a nitrogen/vacuum port. The reactor is
flushed with nitrogen for 15 minutes. To the reactor is charged 2ꢀbutanone (153 kg, 9.0 vol, 7.2
wt), compound 5 (21.1 kg, 1 wt, 1.0 eq) and aluminum isopropoxide (10.36 kg, 0.48 wt, 1.0 eq)
under a nitrogen blanket. To the reactor is charged toluene (164.7 kg, 9.0 vol, 7.8 wt) and
stirring is initiated. The reactor is degassed by first pulling a vacuum until solvent is seen
dripping from the condenser and then back flushed with nitrogen. This process is repeated 4
times. The mixture is then warmed to an internal temp of 80 ± 5 °C and allowed to stir until
completion of the reaction (>12 hours). Once the reaction is deemed complete, the jacket
temperature is set to 45 °C, while simultaneously a solution of potassium sodium tartrate
(Rochelle’s Salt 20%) (300 kg) is added to the vessel. The reaction mixture is allowed to cool to
an internal temperature of 45 ± 5 °C, and stirred for at least 2 hours from the time that the
Rochelle’s Salt solution was added to the reactor. After the hold time is complete, the reaction
mix is cooled to 20 ± 5 °C. The stirring is stopped and the layers are allowed to separate. The
bottom aqueous layer is removed and held in a separate container. The stirring is started, and a
second solution of Rochelle’s Salt 20% (300 kg), is charged to the reactor. The stir rate can be
adjusted so that good mixing of the aqueous and organic phases is achieved. The reaction
mixture is allowed to stir for a minimum of 30 min at an internal temperature of 20 ± 5 °C. The
stirring is stopped and the layers are allowed to separate. The bottom aqueous layer is removed
and held in a separate container. The stirring is started, and toluene (146 kg, 8 vol, 6.92 wt)
followed by water (125 kg, 5.93 vol, 5.93 wt) is charged to the reactor. The stir rate can be
adjusted so that good mixing of the aqueous and organic phases is achieved. The reaction
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mixture is allowed to stir for a minimum of 30 min at an internal temperature of 20 ± 5 °C. The
stirring is stopped and the layers are allowed to separate. The bottom aqueous layer is removed,
while the remaining organic phase (batch) is removed from the reactor and charged to a
receiving vessel. The reactor is cleaned with methanol and blown dry, then the batch is charged
back to the reactor through a polish filter. The stirring is started and to the reactor is charged with
ethanol (83 kg, 5 vol, 3.95 wt). The stir rate can be adjusted to insure vigorous mixing. The
reactor is fitted with an addition funnel, and 31% aqueous citric acid solution (30.8 kg) is
charged to the addition funnel. The reactor containing the batch is degassed by first pulling a
vacuum until solvent is seen dripping from the condenser and then back filled with nitrogen.
This process is repeated 3 times. The contents of the reactor are heated to 45 ± 5 °C. Once the
batch reaches 45 ± 5 °C, approximately 50% of the citric acid solution is charged dropwise (18.5
kg). Once nucleation is observed, stop the addition and age the suspension for 30 ꢀ 45 minutes.
After aging, the remaining citric acid solution (12.3 kg) is charged dropwise. The slurry is aged
at 45 ± 5 °C for a minimum of 1 hour, then cooled to 20 ± 5 °C over an hour, and held at this
temperature for a minimum of 2 hours. The slurry is then heated back to 45 ± 5 °C over the
course of 1 hour and held at this temperature for a minimum of 1 hour. The slurry is cooled back
to 20 ± 5 °C over the course of 1 hour, then held at this temperature for a minimum of 2 hours
before filtration. The solids are filtered, and the wet cake is washed three times with 2ꢀ
methyltetrahydrofuran (48 kg, 1.7 wt, 2 volumes each time). The solid is dried with nitrogen for
1 ꢀ 2 hours until no more solvent is seen exiting the filter, then with warm nitrogen (40 °C) for ≥
12 hours while periodically stirring the filter cake. The solid is dried under vacuum at 40 °C for
≥ 8 hours. This procedure delivers the citrate salt of compound 6. The corresponding free base is
produced by the following method: a size appropriate jacketed reactor is equipped with an
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overhead stirrer, thermocouple, temperature control unit, and a nitrogen/vacuum port is charged
with tetrahydrofuran (107 kg, 2.9 wt, 3.2 volumes), 6ꢀcitrate (36.85 kg, 1 wt, 1 eq), and then
water (6.3 kg, 0.17 wt, 0.17 vol). The mixture is stirred at 20 °C, then a 20 wt% aqueous
potassium carbonate solution (148.9 kg, 4 wt) is charged under a nitrogen blanket. The biphasic
mixture is stirred in the reactor at 30 ± 5 °C for ≥ 30 minutes. At the end of this hold all solids
should be dissolved. After the hold is complete and the internal batch temp is 30 ± 5 °C, the
stirring is stopped and the phases are allowed to separate. The bottom aqueous phase is removed.
To the reactor is charged with another portion of 20 wt% aqueous potassium carbonate solution
(148.9 kg, 4 wt). The batch is allowed to mix at 30 ± 5 °C for ≥ 30 minutes. The stirrer is stopped
and the layers are allowed to separate. The bottom aqueous phase is removed. The batch is
agitated at 20 °C, then ethanol is charged (49, 4 kg, 1.7 vol, 1.32 wt) all at once. Water (348 kg,
10.2 vol, 10.2 wt) is then charged dropwise to the reactor. When nucleation is observed, the
batch is held at 20 ± 5 °C for ≥ 30 minutes. The batch is filtered with 30 micron nylon filter
cloth to collect solids. The reaction and wet cake is rinsed with at least 2ꢀ3 cake volumes of
water (205 kg). The solid is dried with nitrogen for 1 ꢀ 2 hours until no more solvent is seen
exiting the filter. The solid is dried with warm nitrogen (40 °C) for ≥ 12 hours, while periodically
stirring the filter cake. The solid is dried under vacuum at 40 °C for ≥ 8 hours, while periodically
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stirring the filter cake. Mp = 199 °C; H NMR (400 MHz, Pyridineꢀd5) δ 5.89 (d, J = 1.9 Hz,
1H), 4.92 (s, 1H), 3.46 (ddd, J = 10.7, 9.5, 3.9 Hz, 1H), 3.07 (ddd, J = 12.2, 4.2, 1.5 Hz, 1H),
2.77 (dd, J = 9.6, 7.2 Hz, 1H), 2.58 – 2.12 (m, 8H), 2.01 (dddd, J = 28.6, 13.2, 7.5, 2.4 Hz, 5H),
1.90 – 1.61 (m, 8H), 1.53 (dddt, J = 13.7, 10.4, 6.9, 3.4 Hz, 1H), 1.40 (tdd, J = 12.5, 10.0, 3.8 Hz,
2H), 1.22 (q, J = 11.2 Hz, 1H), 1.17 – 1.05 (m, 2H), 1.02 (d, J = 7.3 Hz, 3H), 0.96 (s, 3H), 0.83
(d, J = 6.7 Hz, 3H); 13C NMR (101 MHz, Pyridineꢀd5) δ 198.72, 170.54, 140.42, 125.82, 125.40,
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