Synthesis of vaniprevir (MK-7009): Lactamization to prepare a 22-membered macrocycle

Article abstract of DOI:10.1021/jo2011494

Development of a practical synthesis of MK-7009, a 22-membered macrocycle, is described. A variety of ring-closing strategies were evaluated, including ring-closing metathesis, intermolecular palladium-catalyzed cross-couplings, and macrolactamization. Ri

Full text of DOI:10.1021/jo2011494

ARTICLE
pubs.acs.org/joc
Synthesis of Vaniprevir (MK-7009): Lactamization To Prepare a
22-Membered Macrocycle
Zhiguo J. Song,* David M. Tellers,* Michel Journet, Jeffrey T. Kuethe, David Lieberman, Guy Humphrey,
Fei Zhang, Zhihui Peng, Marjorie S. Waters, Daniel Zewge, Andrew Nolting, Dalian Zhao, Robert A. Reamer,
Peter G. Dormer, Kevin M. Belyk, Ian W. Davies, Paul N. Devine, and David M. Tschaen
Department of Process Chemistry, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, United States
S Supporting Information
b
ABSTRACT: Development of a practical synthesis of MK-7009, a 22-membered macro-
cycle, is described. A variety of ring-closing strategies were evaluated, including ring-closing
metathesis, intermolecular palladium-catalyzed cross-couplings, and macrolactamization.
Ring closure via macrolactamization was found to give the highest yields under relatively high
reaction concentrations. Optimization of the ring formation step and the synthesis of key
intermediates en route to MK-7009 are reported.
’ INTRODUCTION
Hepatitis C virus (HCV) affects an estimated 170 million
people worldwide and is the leading cause for liver trans-
plants.1 Existing standard of care combines Pegylated inter-
feron and ribavirin and only provides a modest cure rate.2 This
low cure rate is partially attributed to the fact that patient
response is highly dependent on the genotype of the virus. To
address this, various gene targets have been evaluated in order
to identify new modes of treatment. Recent regulatory filings of
Figure 1. Macrocyclization strategies to prepare intermediate 9.
boceprevir and telaprevir promise to improve the HCV therapy
success rate. Despite these tremendous accomplishments,
additional improvements in the area of genotype coverage,
’ RESULTS AND DISCUSSION
drug dose, and cure rate are still desired. One potential option
is the HCV NS3/4a protease inhibitor vaniprevir (MK-7009),
a compound in late phase clinical studies (Figure 1).3ꢀ6 In
order to support this promising candidate in preclinical and the
initial clinical studies, a scalable process permitting access to
MK-7009 was required.
Employing the medicinal chemistry synthesis as a starting point
for preparation of MK-7009, we began to evaluate potential methods
for preparing macrocycle 9. From the outset, it was clear that total
route optimization could not be initiated until a cyclization strategy
was identified, as this would define the overall synthetic approach.
Consequently, we first detail our investigation into the identification
of a suitable macrocyclization strategy, and second, we describe the
synthesis and optimization of the premacrocyclization intermediates,
macrocyclization reaction, and final steps to prepare MK-7009.
Ring-Closing Metathesis. While numerous methods for ring
closure can be envisioned, we primarily focused on ring-closing
olefin metathesis (RCM), intramolecular Pd-catalyzed cross
coupling reactions, and macrolactamization (Figure 1). Initial
efforts were placed on examination and optimization of the RCM
route. Despite proceeding in good yields, the ring closure high-
lighted in Scheme 1 required high catalyst loadings and high
dilution conditions (<3 mM). Initial efforts were placed on
optimizing the reaction catalyzed by Neolyst M1 catalyst due
to its easy access and relatively low cost. However, we observed
thatwhen catalystsloading was reduced orwhen the concentration
The synthesis of MK-7009 employed by our colleagues in
the Department of Medicinal Chemistry is illustrated in
Scheme 1. MK-7009 is a 22-membered macrocycle con-
structed from four primary fragments: isoindoline (1), hydro-
xyproline (2), tert-butylglycine linker (5), and a cyclopropyl-
sulfonamide (8). Following amide and carbamate bond for-
mations with 1, 2, and 5, the macrocycle is constructed via a
high dilution ring-closing metathesis reaction catalyzed by
ruthenium compounds.7 Subsequent coupling of the cyclo-
propylsulfonamide (8) with macrocyclic acid yields MK-7009.
After MK-7009 was identified as a clinical candidate, a closer
evaluation of this synthesis was initiated with the goal of
improving efficiency, yield, and productivity to support clin-
ical needs. Herein, we describe the successful development of
a practical synthesis of MK-7009 with particular emphasis
placed on our efforts to develop an efficient ring-closing
strategy.
Received: June 4, 2011
Published: August 12, 2011
r
2011 American Chemical Society
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Scheme 1. Medicinal Chemistry Synthesis of Vaniprevir (MK-7009)
of the reaction was increased, substantially lower yields were
obtained. Rigorous purification of the starting material was
required to get complete conversion, as incomplete reactions
could not be driven to completion by additional catalyst.8 Catalyst
removal after the reaction also posed challenges. In addition to
their material high costs, many RCM catalysts are under patent
protection. At this point, extensive work was needed to make the
process amendable to scale up. Given these challenges, project
timeline and material demands, further development of this
specific strategy was halted and efforts were placed on identifying
a more efficient ring-closing strategy.9ꢀ12
Palladium-Catalyzed Macrocyclization. Given the vast array
of potential cross-coupling reactions and ligands available to effect
these transformations, we opted to examine ring closure via
palladium catalysis.13ꢀ21 The intermediates used to prepare the
RCM substrate wereeasily manipulatedto generate molecules that
could be subjected to palladium-catalyzed macrocyclization con-
ditions. Inparticular, wehave madeintermediates10, 12, and 13in
Scheme 2 and investigated Heck, Sonogashira, and Suzuki
macrocyclizations.22 For the Heck reaction, high-throughput
catalyst, solvent, and base screen showed that most common
conditions produced the desired macrocycle 11. The best assay
yield of the desired product 11 that was verified on 60 mgscale was
47%, which was achieved under high dilution conditions. Compet-
ing oligomerization, dehydrobromination, and 21-membered ring
formation were encountered. For the Suzuki cyclization, we
investigated very briefly the 9-BBN adduct 13 under a few
common reaction conditions. Low reactivity and side reactions
from the substrate 13 led to only a trace amount of product as
detected by LCꢀMS. For the Sonogashira cyclization, even under
high dilution conditions, a substantial amount of dimer was
observed by LCꢀMS and the assay yield of the desired product
14 was only 34%. While continued optimization would likely lead
to improved yields, ring closure via macrolactamization, a route
being pursued in parallel, began to yield better results.
Macrolactamization. Cyclization via lactam formation is a
frequently employed strategy in ring constructions.23ꢀ28 As such,
we investigated closing the macrocycle at proline and tert-
butylglycine position (eq 1). Prior to investing efforts to optimize
the synthesis of the precursors for macrolactamization, inter-
mediate 16 was prepared on a small scale using a modification of
the medicinal chemistry protocol. We were pleased to find that
use of EDC with HOBt in DMF facilitated conversion to 9 in
purity and yield superior to those obtained with the RCM and
Pd-catalyzed cyclizations. Importantly, this scouting reaction
served as proof of the effectiveness of the macrolactamization
approach. Subsequent emphasis was therefore placed both on
optimizing the macrocyclization reaction and on preparation of
the macrolactamization precursors 5, 15, and 16 (eq 1).
Synthesis of the Heck Coupling Partners 5 and 15. As
shown in eq 1, coupling of intermediates 5 and 15 was viewed as
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Scheme 2. Macrocycle Formation via Heck, Suzuki, and Sonogashira Reactions
Scheme 3. Optimized Process for Synthesis of Intermediate 15
the route to premacrocycle 16. While alternate methods for
connecting these two fragments are possible (vide infra), we
initially focused on generating this bond through a Heck reaction
and subsequent hydrogenation. The chemistry to prepare inter-
mediates 5 and 15 involved modification and optimization of the
medicinal chemistry precursors, as shown in Schemes 3 and 4.
Notable changes in the synthesis of N-benzylisoindolene 18
include replacement of carbon tetrachloride with chlorobenzene
in the bromination reaction and crystallization of the tosylate salt
directly from the cyclization reaction in contrast to distillation of
the corresponding free base. Subsequent debenzylation followed
by CDI-mediated carbamate formation with Cbz-protected
hydroxy proline ester gives 15, which was isolated directly from
DMF/water by crystallization. The Cbz protecting group
was used in place of the Boc group to streamline the synthesis
as hydrogenation of the macrolactamization precursor
would both reduce the Heck alkene product and cleave the
Cbz group.
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The tert-butylglycine derived linker 5 was prepared in three
steps with one isolation (Scheme 4). Low-temperature deproto-
nation and alkylation of ethyl isobutyrate produced the desired
ester 20 in good assay yield. Following aqueous workup, this
crude solution was taken into a DIBAL-H reduction to generate
alcohol 21, which was then treated with CDI and tert-butylgly-
cine to produce 5, which was isolated as the dicyclohexylamine
salt (5-DCHA).
sources catalyzed formation of 22 (Scheme 5). In general, these
reactions proceeded in greaterthan >85% assay yield ofthe desired
linear olefin product and typically 10ꢀ12% of the undesired
nonlinear olefin regioisomers (22-exo) regardless of the ligand
identity.29 Despite an exhaustive ligand and solvent screen, the
ratio of regioisomers could not be changed, suggesting that the
reactive catalyst either does not contain a ligand or that the
regiochemistry-determining step is not influenced by ligand.30
Intrigued by the observation that nearly every ligand gave the same
yield and regioisomer ratio, Pd(OAc)2 without added phosphine
ligand was examined for the cross-coupling reaction. In this case,
similar yields and regioisomer ratios were obtained without ligand.
This suggests that productive bond formation is likely going
through ligandless palladium, thereby explaining the results from
our initial screen where ligand was found to have minimal impact
on yield or selectivity. Following additional screening, we deter-
mined that Pd(OAc)2 loaded as low as 0.75 mol % in NMP
catalyzedformationof22inidentical yieldand regioisomerratioto
the reactions optimized with ligand.31ꢀ33
Heck Reaction and Hydrogenation. We were pleased to find
that a variety of ligands in combination with Pd(II) and Pd(0)
Scheme 4. Optimized Synthesis of Olefin Linker 5
The desired Heck product was not isolated from the reaction
mixture but taken directly into the subsequent hydrogenation
(Scheme 5). In most cases, the residual palladium from the Heck
reaction was sufficient to catalyze hydrogenation of Heck reac-
tion products; however, conversions typically stalled at ∼95% by
HPLC (area %). It was deemed critical to take the hydrogenation
to >99% conversion to control process impurities downstream
(vide infra). Therefore, in order to ensure acceptable conversion
(>99%), a 5% by weight charge of 10% Pd/C was added prior to
introduction of hydrogen. With this added catalyst charge, the
hydrogenation was typically complete (g99.8% conversion)
in <12 h. Attempts to crystallize the premacrocyclization
Scheme 5. Preparation of Macrolactamization Precursors via Heck and Hydrogenation Reactions
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Figure 2. Selected list of coupling agents examined in the macrolactamization reaction.
intermediate 16 were unsuccessful. We thus opted to take the
crude reaction mixture directly into the macrocyclization, which
would require removal of Heck, hydrogenation, and macrocycli-
zation impurities after the cyclization reaction.
temperature improved the reaction rate at the expense of assay
yield. Overall, the HATU-mediated cyclization proved to be a
superior reagent in terms of reaction rate and yield. However, the
combination of EDC/HOPO provided a cost-effective alterna-
tive to HATU. Consequently, we opted to examine both the
HATU and EDC/HOPO leads in greater detail with particular
emphasis placed on optimizing yield while reaction concentra-
tion was increased and purification and isolation strategies were
identified.
HATU-Mediated Cyclization. With HATU, we first sought
to understand the impact of dilution on reaction yield. Increas-
ing reaction concentration above 20 g/L results in a precipitous
drop in yield with concomitant formation of dimer, trimer, and
additional higher order species. The best yields were obtained
with substrate concentration below 10 g/L of substrate; how-
ever, little additional yield improvement was realized below
20 g/L. One potential strategy for overcoming this high dilution
requirement would be to mimic the high dilution conditions by
slow addition of the macrocycle precursor 16 to a concentrated
solution of the coupling agent. For this to be successful, the rate
of addition has to be equal to or less than the rate of macro-
cyclization to prevent buildup of 16. Fortunately, the reaction
with HATU is instantaneous at all temperatures examined.
After careful examination of reaction rate, temperature, and
concentration, we were pleased to find that reverse addition
reactions run at final substrate concentration of 50 g/L instead
of 10 g/L produced the desired macrocycle 9 in yields
(70ꢀ75%) similar to the high dilution conditions with a nearly
identical impurity profile.
Macrolactamization Optimization. Initial priority was placed
on identifying an optimum amide bond forming reagent. This
screening was done under high dilution conditions to minimize
formation of oligomeric products. In particular, the hydrogena-
tion reaction mixture which was in NMP solvent was diluted
with different solvents to obtain a final concentration of 10 g/L
(16/solvent).8 A selection of the amide bond forming reagents
examined is shown in Figure 2 and were either uronium-derived
reagents or a mixture of EDC with an in situ activating agent.34
Complete conversion of starting material was observed in nearly
all cases; however, the corresponding assay yield and reaction
time varied dramatically with the identity of the coupling reagent.
In general, the uronium-derived reagents gave higher yields and
shorter reaction times compared to the EDC derived reagents.
For example, HATU gave the highest assay yield within a very
short time frame (100% conversion, 75% assay yield, < 5 min).35
The EDC-mediated cyclization, in particular with HOPO,36 also
produced 9 in good yield; however, reaction times were sig-
nificantly longer (∼12 h). Note that the assay yield reported is of
the desired macrocycle and does not include the contribu-
tion from regioisomer cyclization (∼10% assay yield).37 The
remaining 10% is a mixture of dimeric, trimeric, and oligomeric
macrocycles, which were identified by mass spectrometry.
Reaction temperature and solvents were also examined. In
general, strongly polar aprotic solvents, like DMF, gave the best
yields, while acetonitrile and THF could also be used with
approximately a 10% reduction in yield. Increasing the reaction
EDC/HOPO Macrolactamization. It should be noted that
HATU is a relatively expensive coupling agent. As noted
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Scheme 6. Final Steps To Prepare MK-7009
above, we also identified EDC/HOPO as an effective surro-
gate for HATU. Analogous to the reaction with HATU, the
EDC/HOPO-mediated cyclization is very sensitive to reac-
tion concentration, with optimum yields obtained at or below
10 g/L. For EDC/HOPO, reactions were typically complete
within 12 h. The rate-limiting step in this cyclization is likely
ring closure of the proline amine with the HOPO-activated ester
of the tert-butylglycine intermediate. This intermediate was
observed by HPLC and NMR and was formed within ∼30 min
after addition of EDC to the reaction mixture (eq 2).
addition of water. This served to remove the majority of the
palladium waste, HATU- or EDC/HOPO-derived byproduct, the
remaining DCHA salt, and, most importantly, nearly all of the
regioisomers (exomethyl isomer) that originated from the Heck
reaction. Unfortunately, the dimer/trimeric/oligomeric species
were not removed. Instead of carrying this mixture forward for
downstream purification, these impurities were removed by
redissolving the macrocycle 9 in isopropyl acetate with 5 mol
equiv of water and subsequently crystallized by slow addition of
methylcyclohexane to produce the macrocycle with <1% dimer/
trimer/oligomeric impurities as the crystalline monohydrate.
Thus, the 22-membered macrocycle 9 was obtained in sufficient
purity after three steps and two crystallizations in 50ꢀ55% overall
yield from intermediate 15.
Final Coupling and API Isolation. Preparation of MK-7009 is
illustrated in Scheme 6. Saponification of the ester intermediate
(compound 9) was straightforward, providing the desired acid in
nearly quantitative yield (98% assay yield). Removal of color
generated during the saponification process was accomplished by
treatment with Darco KB-G (40 wt %). The acid was not isolated,
the solvent was switched from IPAc (isopropyl acetate) to DMF
prior to side chain coupling. Formation of MK-7009 is accom-
plished in 91% assay yield by treating a DMF/IPAc solution of
the macrocyclic acid with the side chain 8,38,39 EDC, HOPO, and
DIPEA. Following an aqueous workup, the isopropyl acetate was
replaced with ethanol via constant volume distillation. Careful
addition of aqueous potassium hydroxide produces the potas-
sium salt, which crystallizes from solution. This crystalline
potassium salt is a nonstoichiometric hydrate and the water
content depends on humidity of the storage conditions. The
drying process also needs to run under nitrogen with controlled
humidity. Alternatively, the neutral MK-7009 can be isolated
from IPAcꢀheptane as a heptane solvate, which upon drying
turns into anhydrous crystalline solid. Both forms were used in
clinical formulations.
Additional Avenues for Route Optimization: Sonogashira
Route. With a robust process in hand to support preparation of
MK-7009 for clinical needs, we again shifted our attention to
improving the overall yield of the macrolactamization route. In
particular, we focused on identifying alternatives to the Heck
reaction, as it resulted in a 13% yield loss due to regioisomer
formation. We addressed this challenge by focusing on the use of
a Sonogashira-type coupling, which would remediate the regioi-
somer issue.40ꢀ42 The preparation of the alkyne linker is
illustrated in Scheme 7 and relies upon a key “zipper” reaction
to convert an internal alkyne 24 to the required terminal alkyne
25. Subsequent carbamate formation with CDI and tert-butyl-
glycine followed by treatment with DCHA yields the salt of
Efforts to expedite this cyclization were unsuccessful. Conse-
quently, attempts to run this reaction using the reverse addition
strategy gave lower yields depite extensive exploration. In order
to maximize yields, we therefore ran the cyclization reaction at
10 g/L substrate in a 50/50 mixture of DMF/acetonitrile.
Following reaction completion, the acetonitrile is removed under
reduced pressure to produce a 20 g/L solution of 16. Increasing
the acetonitrile concentration beyond 50% resulted in an un-
acceptable loss of yield. Given the cost of HATU, we ultimately
opted to use the EDC/HOPO reaction conditions to support
ongoing trials.
IsolationofMacrocycle9. Havingidentifieda macrocylization
reaction, emphasis shifted toward isolation of macrocyle 9 in
acceptable purity. This would require removal of impurities
generated during the Heck, hydrogenation, and cyclization reac-
tions. Specifically, we were concerned about removal of regioiso-
meric impurities and dimeric/trimeric impurities, which would be
difficult given their similarity to the desired product. After careful
screening of conditions, it was determined that crystallization of 9
from the DMF reaction mixture could be effected by slow
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Scheme 7. Sonogashira and Hydrogenation Sequence To Produce Premacrocycle 16
alkyne linker 26. The Sonogashira reaction of 26 and aryl
bromide 15 was mediated by the X-Phos/Pd catalyst.43 In
contrast to the Heck reaction, this Sonogashira coupling step
proceeds in near quantitative yield without production of multi-
ple isomers. Subsequent hydrogenation of this coupling product
yields intermediate 16, which converges with the current macro-
lactamization route.44
The overall relative yield of the Sonogashira through macro-
lactimization is 10ꢀ15% higher than the Heck route due to the
absence of regioisomers generated in the Heck reaction. How-
ever, the starting material cost required for the alkyne synthesis is
substantially higher due to the added cost of 1-bromo-2-butyne
and butyllithium. While the Heck reaction produces 13% of the
undesired regioisomer, it employs ligandless conditions with
palladium, while the Sonogashira reaction requires use of a
relatively expensive ligand. In addition, this route requires one
additional synthetic transformation. Taken together, the Heck
route was viewed as both more robust and cost-effective than the
Sonogashira route to MK-7009 at this stage in our development.
Nonetheless, the Sonogashira route will be attractive if an
alternate synthesis of the alkyne precursor is identified in concert
with a more cost-effective Sonogashira coupling reaction.
MHz spectrometers. Low-resolution mass spectra were taken by
LCꢀMS with acetonitrile and ammonium formate buffer and the mass
spectrometer set for positive electron ionization. The weight purity of
isolated compound or crude solution may be analyzed against a working
standard and reported as weight percent. The purity or reaction
conversion based on HPLC peak integration may also be reported as
area percent (A%).
3-Bromo-N-benzylisoindoline pTSA Salt (18-PTSA). 3-Bromo-
O-xylene (17.1 g, 94.6 mmol) and N-bromosuccinimide (36.1 g, 208.1
mmol) were charged to a round-bottom flask followed by chlorobenzene
(194 g) and benzoyl peroxide (131.3 mg, 0.38 mmol). The mixture was
stirred and warmed to 88ꢀ90 °C for 1.5 h and another charge of benzoyl
peroxide (131.3 mg, 0.38 mmol) was made. The mixture was stirred at
88ꢀ90 °C for a further 2 h, and HPLC indicated complete reaction. The
batch was cooled to 25 °C and washed with water (3 ꢁ 75 mL), giving the
dibrominated product 17 (estimated 23.8 g, 75% yield) as a solution
in chlorobenzene. The chlorobenzene solution was diluted with toluene
(147 g) and water (3.0 mL) added. Solid sodium bicarbonate (14.2 g, 173.8
mmol) was charged to the vessel followed by benzylamine (10.0 g, 93.7
mmol). The reaction was heated to 93ꢀ95 °C for 1.5 h, when HPLC
indicated complete reaction (Caution: carbon dioxide off gas!). The batch
was cooled to 20 °C and washed with water (140 g) and then the organic
layer extracted in to 1 M phosphoric acid (3 ꢁ 100 g). The lower aqueous
extracts were combined and returned to the clean vessel. p-Toluenesulfonic
acid monohydrate (2.4 g) was charged as a solid to the batch and the
mixture seeded. The batch was stirred at 20 °C for 20 min for seed bed
to form before adding the remaining p-toluenesulfonic acid monohydrate
(9.6 g, 63.1 mmol total, 1.2 equiv based on assay) in one portion. The
mixture was stirred at 20 °C for 1 h before filtering. The solid was washed
with water (50 g), collected on trays, and dried in vacuum at 40 °C
for 3 days. 3-Bromo-N-benzylisoindoline pTSA salt 18-PTSA (19.98 g)
was isolated as an off-white solid in 47% yield from 3-bromo-o-xylene, 99%
’ SUMMARY AND CONCLUSIONS
The primary challenges to developing a scalable, cost-effective
synthesis for MK-7009 are identifying an efficient ring-closing
strategy and a concise synthesis of the intermediates needed to
construct the macrocycle precursors. The choice of Heck reac-
tion followed by macrolactamization was based on the overall
efficiency and cost of the process for all the steps. Taken together,
we were able to develop a scalable route to MK-7009 in ca. 20%
overall yield to support clinical trials.
1
pure based on HPLC analysis. Mp 187ꢀ189 °C; H NMR (400 MHz,
CD3OD) δ 7.64 (m, 2H), 7.60ꢀ7.58 (om, 2H), 7.55 (m, 1H), 7.52ꢀ7.49
(om, 3H), 7.35ꢀ7.29 (om, 2H), 7.19 (m, 2H), 4.78 (s, 2H), 4.68 (s, 2H),
4.65 (s, 2H), 2.35 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 143.6, 141.8,
137.1, 135.7, 133.30, 132.4, 131.7, 131.5, 130.7, 130.0, 127.0, 123.5, 118.0,
60.4, 59.6, 21.4; EIMS M + 1 = 288, 290 calcd for C15H14BrN = 287, 289.
’ EXPERIMENTAL SECTION
General. Chemical reagents were used as received from suppliers
except otherwise noted. NMR spectra were taken on 400, 500, or 600
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Anal. Calcd for C22H22BrNO3S: C, 57.39; H, 4.82; N, 3.04; S, 6.96. Found:
C, 57.53; H, 4.70, N, 2.97, S, 6.70.
53.51, 53,.16, 53.11, 52.83, 52.73, 52.62, 52.36, 37.24, 37.14, 36.18,
36.09; [α]25859 = ꢀ46 (1 g/100 mL MeOH); EIMS M + 1 = 503, 505,
calcd for C23H23BrN2O6 = 502, 504. Anal. Calcd for C23H23BrN2O6: C,
54.88; H, 4.61; N, 5.57. Found C, 54.99, H, 4.37, N, 5.47.
3-Bromoisoindoline HCl Salt (19). 3-Bromo-N-benzylisoindo-
line pTSA salt 18-PTSA (40.5 g, 87.9 mmol) was charged to a 500 mL
round-bottom flask followed by chlorobenzene (179 g). Sodium hydro-
xide solution (1 M, 200 mL) was charged to the flask in one portion and
the mixture stirred vigorously for 15 min until all the solids had
dissolved. The layers were separated, and the bottom organic layer
was washed with water (132 g). Once again the layers were separated,
and the bottom organic layer was azeotropically dried by distilling
chlorobenzene (20 mL) from the mixture under reduced pressure (KF
of batch = 51 μg/mL).
2,2-Dimethyl-5-hexenol (21). Alkylation. A 50 mL round-bot-
tom flaskwas chargedwith THF(10 mL) and diisopropylamine (3.25 mL,
2.35 g, 23.2 mmol) and the solution cooled to around ꢀ20 °C. Hex-
yllithium (2.3 M in hexane, 9.65 mL, 22.2 mmol) was added over 30 min
at ꢀ20 to ꢀ10 °C, and the batch was aged for an additional 15 min. Ethyl
isobutyrate (2.46 g, 21.1 mmol) was added over 15ꢀ30 min while the
batch temperature was kept between ꢀ10 to ꢀ20 °C. At the end of
the addition, DMPU (1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidi-
none, 2.53 mL, 21.1 mmol) was added over a few minutes, and the
resulting solution was aged at ꢀ10 to ꢀ20 °C for 15 min. 4-Bromo-1-
butene (3.0 g, 22.2 mmol) was then added dropwise over 15ꢀ30 min
while keeping the batch temperature around ꢀ20 °C. The resulting thin
slurry was aged for 30 min at ꢀ20 °C, allowed to warm to rt, aged
for another hour, and quenched by MTBE (21 mL) and aqueous HCl
(1.5 N, 17 L, 25.3 mmol). Layers were separated, and the organic
layer was washed with water (2 ꢁ 17 mL) and concentrated to an oil
(3.31 g assay, 92% yield of 20), which was dissolved in toluene (6 mL),
and concentrated again to give 4.85 g of crude liquid product 20
(65 wt %, 3.16 g assay, 88% isolated yield), which was used in the next
step without further purification (Caution: the product is volatile; do not
overdistill). 1H NMR spectroscopy was used to confirm the identity of
intermediate 20.3
DiBAL-H Reduction. A round-bottom flask was charged with a 1 M
THF solution of diisobutylaluminum hydride (DiBAL-H, 30.7 mL,
30.7 mmol) and was cooled to around ꢀ20 °C. Crude ester 20 (2.9 g,
75 wt %, 2.18 g assay, 12.8 mmol) was added over 30 min and the
temperature was kept around ꢀ10 °C. The batch was aged for 30 min
and was transferred over 30 min into a biphasic solution made of MTBE
(22 mL) and a 1.5 M aqueous Rochelle’s salts solution at 0 °C. The
resulting mixture was aged at 5ꢀ10 °C for 1 h, allowed to warm to rt, and
aged 2 h. Layers were separated, and the organic layer was washed with
1 N aqueous HCl (17 mL, 17 mmol) and with water (2 ꢁ 17 mL) and
concentrated to give 3.2 g of crude oily product 21 (51 wt %, 1.64 g assay,
100% assay yield), which was used in the next step without further
purification. NMR spectroscopy was used to confirm the identity of the
material.3
1-Chloroethylchloroformate (ACE-Cl, 16.3 g, 114 mmol) was added
to the above solution keeping the batch temperature <15 °C. The batch
was warmed to 90ꢀ95 °C and stirred at this temperature for 2 h. HPLC
analysis indicated 8.8% starting material remaining. More 1-chloroethyl-
chloroformate (1.14 g, total 122 mmol) was added to the reaction and the
batch heated to 90ꢀ95 °C overnight (14 h). HPLC analysis indicated the
reaction was complete, with <2% starting material remaining. The batch
was cooled to 40 °C and methanol (32 g) charged to the flask. The
mixture was heated to 60ꢀ65 °C and kept at this temperature for 4 h.
HPLC analysis indicated the reaction was complete, with <1% carbamate
intermediate remaining. The batch was cooled to 20 °C and aged for 1 h
beforefiltering. The solidwaswashedwithacetonitrile(76g), collectedon
trays, and dried in vacuo at 40 °C for 4 days. 3-Bromoisoindoline HCl salt
19(18.4g) was isolatedasawhite solidin 89% yield, 98.2%purebyweight
1
based on HPLC analysis. Mp 326ꢀ328 °C; H NMR (400 MHz,
DMSO-d6) δ 10.19 (br s, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8
Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 4.62 (s, 2H), 4.47 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 137.4, 135.5, 131.0, 130.6, 122.3, 116.30,
51.0, 51.0; EIMS M + 1 = 198, 200, calcd for C8H8BrN = 197, 199.
Anal. Calcd for C8H8BrNHCl: C, 40.97; H, 3.87; N, 5.97. Found C,
41.16; H, 3.63, N, 5.86.
CBZ Carbamate Bromide (15). To a 3 L, four-neck round-
bottom flask, with mechanical stirrer, thermocouple, and nitrogen/
vacuum line, was charged DMF (190 mL), N-Cbz-L-hydroxyproline
methyl ester (54.3 g, 194.5 mmol) and 1,10-carbonyl diimidazole (30.4 g,
187.5 mmol). The reaction was inerted with vacuum/nitrogen cycle and
heated to 65ꢀ70 °C. After 1 h, HPLC analysis showed 97% conversion
to the intermediate. Bromoindoline HCl 19 (41.4 g, 176.6 mmol) was
added to the reaction mixture and aging at 65ꢀ70 °C continued. The
batch changed from slurry to solution as the reaction proceeded. After 6
h HPLC analysis showed >99% conversion. Then 190 mL of MeCN and
380 mL of water were added sequentially to crystallize the product. The
mixture was seeded after 60 mL of water and aged to develop a seed bed.
The water addition after seeding was done very slowly in order to allow a
good seed bed to form. During the addition the slurry became very thick
and vigorous stirring was necessary. The solid was isolated by filtration.
The cake was washed with 1:1 DMF/water (200 mL) and then water
(2 ꢁ 200 mL). The cake was dried on the filter under nitrogen and then
for 6 h in the vacuum oven at 50 °C. The product was obtained as a pale
pink solid (80.60 g) in 91% yield. HPLC showed >95% by area; chiral
assay >99% ee; mp 99ꢀ100 °C; 1H NMR (500 MHz, CDCl3) mixture of
rotamers δ 7.42 (d, J = 7.7 Hz, 2H), 7.37ꢀ7.31 (om, 4H), 7.27 (m, 1H),
7.22ꢀ7.15 (om, 2H), 5.35 (m, 1H), 5.24 (dd, J = 12.4, 7.1 Hz, 0.53H),
5.20 (d, J = 12.4 Hz, 0.47H), 5.14 (dd, J = 12.4, 5.3 Hz, 0.53H), 5.07 (d,
J = 12.4 Hz, 0.47H), 4.81 (br s, 1H), 4.73ꢀ4.68 (om, 2H), 3.91ꢀ3.77
(om, 1H), 3.790 and 3.786 (2s, 1.6H), 3.571 and 3.567 (2s, 1.4H),
2.57ꢀ2.47 (om, 1H); 13C NMR (125 MHz, CDCl3) ∼1:1:1:1 mixture
of rotamers δ 172.82, 172.78, 172.65, 172.60, 155.05, 155.01, 154.40,
153.88, 153.80, 138.44, 138.29, 138.23, 137.57, 137.44, 137.40, 136.50,
136.42, 130.78, 130.74, 129.65, 129.61, 129.58, 128.61, 128.60, 128.23,
128.22, 128.07, 121.73, 121.70, 121.58, 121.55, 117.77, 117.62, 117.58,
73.85, 73.81, 73.16, 73.09, 67.50, 58.22, 58.19, 57.97, 57.94, 54.05, 53.62,
Linker DCHA Salt (5-DCHA). A 50 mL round-bottom flask was
charged with DMF (13 mL) and the crude alcohol 21 (3.2 g, 51 wt %,
1.64 wt %, 12.8 mmol), and the solution was cooled to around 5 °C. CDI
(1,10-carbonyldiimidazole, 2.67 g, 16.5 mmol) was added portionwise
over 15 min. The resulting homogeneous mixture was aged at rt for 30
min. A first portion of CDI (2.08 g, 12.8 mmol) was added, and the
1
reaction was checked by H NMR (CH2O CDI adduct/CH2OH δ
4.1 and 3.25 ppm). More CDI was added until the δ 3.25 ppm peak
disappeared. The reaction was exothermic and the temperature rose to
20ꢀ30 °C over 15 min. L-tert-Leucine [(S)-tert-butylglycine, 2.16 g,
16.5 mmol] was added to the reaction mixture in one portion followed
by the addition of triethylamine (2.5 mL, 17.9 mmol). The resulting
slurry was heated to 90 °C for 12 h and allowed to cool to rt. The slurry
turned homogeneous at 90 °C upon aging. The solution was partitioned
between MTBE (15 mL) and a 0.5 N aqueous NaOH solution (19 mL).
Layers were separated, and the organic phase was discarded. To the
DMF aqueous basic layer was added MTBE (24 mL) and it was
neutralized to pH∼1ꢀ2 with 6 N aqueous HCl solution (ca. 11 mL).
Layers were separated, and the organic layer was washed with with water
(2 ꢁ 15 mL). The organic solution was concentrated, switched to
acetonitrile (ca. 50 mL final, typically KF ∼ 500 ppm), and heated to
45 °C. Dicyclohexylamine (2.32 g, 128 mmol) was added over 1 h. The
salt crystallized, the slurry was aged at 45 °C for 6 h, and the slurry was
allowed to cool to rt, aged 1ꢀ2 h, filtered, and rinsed with acetonitrile
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ARTICLE
(10 mL). The resulting white solid 5-DCHAwas driedat 40 °C inanoven
for 48 h to give 5.1 g of product (85% overall yield). HPLC analysis >90%
by area; chiral HPLC analysis 99% ee; mp 108ꢀ110 °C; 1H NMR (500
MHz, CD3OD) δ 5.81 (ddt, J = 17.0, 10.4, 6.7 Hz, 1H), 4.99 (dt, J = 17.0,
1.6 Hz, 1H), 4.90 (m, 1H), 3.88 (s, 1H), 3.82 (d, J = 10.5 Hz, 1H), 3.73 (d,
J = 10.5 Hz, 1H), 3.17 (dt, J = 11.3, 4.0 Hz, 2H), 2.08ꢀ2.01 (om, 6H),
1.87 (m, 4H), 1.72 (m, 2H), 1.43ꢀ1.29 (om, 10H), 1.22 (m, 2H), 0.98 (s,
9H), 0.933 (s, 3H), 0.929 (s, 3H); 13C NMR (125 MHz, CD3OD)
to form. Additional water (1.95 kg, 70% total charge) was added to the
batch over 5 h. The batch was allowed to stir overnight (16 h). The slurry
was filtered and the filter cake was rinsed with water (500 g ꢁ 3). The
resulting wet cake was dried under vacuum with nitrogen sweep for 20 h.
Crude 9 was obtained as a light tan solid (53.6 g, 67 wt %, 66% isolated
yield for this step, 61% from Heck step). Typically by HPLC analysis,
macrocyclic ester 9 was 95%, dimer 4%, exomethyl isomer 0.5%. Two
diasteromers derived from either (R)-tert-butylglycine or from (2S,4S)-
hydroxyproline were synthesized independently and shown by HPLC
not to be present in the reaction mixture.
δ 177.9, 158.7, 140.6, 114.6, 73.7, 66.4, 54.6, 39.7, 35.4, 35.1, 30.8, 29.6,
25
589
27.7, 26.3, 25.7, 24.8, 24.7. [α] = ꢀ7.22 (1 g/100 mL MeOH); EIMS
M + 1 = 286, calcd for C15H27NO4 = 285. Anal. Calcd for C27H50N2O4:
C, 69.49; H, 10.80; N, 6.00. Found C, 69.49; H, 10.84; N, 5.98
Purification of Macrocyclic Ester 9. The crude ester 9 (25.3 g,
67 wt %) was slurried in IPAc (280 mL) and warmed to 45 °C. After 15
min, the homogeneous solution was mixed with DARCO-KB-G (1.6 g).
After 40 min, the mixture was filtered through a thin layer of Solka Flock.
The filter cake was rinsed with IPAc (33 mL ꢁ 3). Note that the third
rinse with IPAc contained negligible quantities of macrocycle 9.
Quantitative assay indicated that the loss to the carbon treatment is
<5%. The combined IPAc layers were concentrated to 189 mg of 9/g of
solution. The solution was warmed to 50 °C, and water (1.25 mL, 2.25
equiv) was added. After 10 min, previously generated seed (85 mg,
0.5 wt %) was added to yield a thin crystalline seed bed. After 30 min,
methylcyclohexane (470 mL) was added over 1.5 h and the batch was
allowed to cool to room temperature over approximately 2 h. After an
overnight age, the solution was filtered and the light brown filter cake
was washed with MeCy/IPAc (98:2 MeCy/IPAc 50 mL) and dried
overnight under vacuum with a nitrogen sweep. The title compound was
obtained as light brown solid (15.2 g, 96 wt %, water content 3%, 87%
recovery). Typical HPLC area 98% with <0.9% dimer, 0.1% exomethyl
isomer. Typical palladium level was 100 ppm. Overall yield (Heck,
hydrogenation, macrocyclization, recrystallization) to macrocyclic ester
9 hydrate was 53.5%. This yield takes into account measured samples
taken for analysis and front runs. Mp 160ꢀ162 °C; 1H NMR (400 MHz,
CDCl3) δ 7.21 (t, J = 7.5 Hz, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.05 (d, J =
7.5 Hz, 1H), 5.53 (d, J = 9.7 Hz, 1H), 5.32 (t, J = 3.8 Hz, 1H), 4.72 (AB
doublet, J = 15.0 Hz, 2H), 4.63 (dd, J = 10.3, 7.7 Hz, 1H), 4.52 (AB
doublet, J = 15.3 Hz, 2H), 4.42 (t, J = 9.4 Hz, 2H), 4.15 (dd, J = 11.7, 1.7
Hz, 1H), 3.81 (dd, J = 11.7, 3.8 Hz, 1H), 3.76 (s, 3H), 3.24 (d, J = 10.7
Hz, 1H), 2.74 (ddd, J = 14.4, 7.6, 1.4 Hz, 1H), 2.52 (ddd, J = 13.3, 10.1,
6.2 Hz, 1H), 2.41 (ddd, J = 13.3, 10.7, 6.2 Hz, 1H), 2.15 (ddd, J = 14.3,
10.5, 3.9 Hz, 1H), 1.77 (s, 1H), 1.49 (m, 1H), 1.34ꢀ1.25 (om, 3H), 1.16
(m, 1H), 1.06 (s, 9H), 0.96 (s, 3H), 0.79 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 172.3, 171.0, 156.5, 153.8, 137.8, 136.4, 135.2, 128.2, 127.8,
Heck Reaction To Produce 22 and 22-exo. A three-neck
1000 mL round-bottom flask was charged with bromide 15 (50.0 g,
99.3 mmol), 5-DCHA (55.6 g, 120 mmol, 1.2 equiv), NMP (75 mL).
The heterogeneous mixture was stirred and warmed to 45 °C. Upon
dissolution of all solids, the red-orange solution was subjected to
nitrogenꢀvacuum purge cycles and placed under nitrogen (Degassing
at room temperature results in formation of an intractable foam). The
catalyst, palladium acetate (0.223 g, 1.0 mmol), was weighed out in the
air and quickly transferred to the reaction flask. After the catalyst charge,
the solution was subjected to three nitrogenꢀvacuum purge cycles,
placed under nitrogen, and warmed to 100 °C. The reaction solution
darkens over time, after which a black precipitate forms. Qualitatively,
this typically coincided with the end of the reaction (1.5 h). After 2 h, the
solution was sampled and judged to be complete by HPLC (>99%
conversion by area). Upon cooling to rt, the heterogeneous reaction was
charged with BHT (butylated hydroxyltoluene, 0.25 g, 1.1 mmol, to
inhibit air oxidation) and transferred to the hydrogenation vessel. The
original reaction vessel was rinsed with NMP (10 mL ꢁ 2) and the NMP
was combined with the reaction solution in the hydrogenation vessel.
White solid (DCHA HBr salt) is present in the mixture.
3
Hydrogenation To Produce 16 and 16-exo. To the solution of
Heck product 22 and 22-exo (97.9 mmol, in NMP as described in the
previous step) was added dry palladium on carbon (Johnson Matthey, dry
10%, 2.5 g). The solution was transferred to a hydrogenation vessel,
subjected to five vacuumꢀnitrogen purge cycles, and placed under hydro-
gen gas (40 psig) at room temperature. After overnight aging, the solution
was vented to atmospheric pressure and placed under nitrogen. The black
solution was filtered through a thin layer of Solka Floc and the filter cake was
rinsed with DMF (1000 mL). The DMF/NMP solution of product was
carried on to the macrocyclization. HPLC indicated the ratio of the 16 to
16-exo product was typically 88:12. The final target concentration after
filtration and DMF rinse was approximately 0.064 mmol/g.
120.3, 74.4, 72.7, 59.4, 57.8, 54.3, 52.8, 52.5, 51.1, 37.1, 36.8, 35.7, 34.3,
25
589
33.8, 30.9, 26.4, 25.3, 24.0, 23.6; [α]
= ꢀ64.05 (0.5 g/100 mL
Macrocyclization Reaction with EDC/HOPO To Produce
Macrocyclic Ester 9 and 9-exo. At 20 °C, the DMF/NMP solution
of hydrogenation product 16 and 16-exo (98 mmol total isomers in
88/12 ratio in 1.505 kg solution) was added to a 12 L flask containing
HOPO (14.5 g, 130.4 mmol). DMF (1.5 L) and acetonitrile (2.8 L)
were added. After 10 min, DIPEA (diidopropylethylamine, 30.3 mL,
MeOH); EIMS M + 1 = 558, calcd for C30H43N3O7 = 557. Anal. Calcd
for C30H43N3O7H2O: C, 62.59; H, 7.88; N, 7.30. Found C, 62.73; H,
7.77; N, 7.32.
Macrocyclization via Reverse Addition To Produce Macro-
cyclic Ester 9 and 9-exo. HATU (9.93 g, 26.1 mmol) followed by
DMF (147 g) were charged to a 500 mL round-bottom flask. The
mixture was stirred to dissolve the solid and then cooled to 0 °C. The
compound 16 DMF solution (from 21.3 mmol aryl bromide Heck
reaction followed by hydrogenation, 12.1 g estimated desired isomer)
was mixed in another 500 mL flask together with DIPEA (12.8 g,
94.6 mmol) at <30 °C. This basic solution was slowly added to the
HATU/DMF solution over 2 h at 0 °C and the mixture stirred at 0 °C for
an additional 30 min. HPLC indicated <0.5% starting amino acid
intermediate remaining. The batch was warmed to 20 °C. Water (80 g
total) was charged to the batch and the temperature was kept at
20ꢀ25 °C, producing a cloudy mixture. The batch was seeded and
stirred for 20 min before resuming the water addition (162 g over 2 h).
The batch was stirred at 20 °C overnight and filtered, and the cake was
washed with water twice (25 g each). The solid was dried under nitrogen
on the filter for 3 days and then transferred to the oven and dried in
174.0 mmol) was added to the stirred solution followed by EDC HCl
3
(33.3 g, 174.0 mmol). The mixture was stirred at room temperature
overnight. The reaction was judged complete by HPLC (>99% con-
version). During the aging, some DCHA HCl crystallized from solu-
3
tion. HPLC indicated that the macrocyclic ester concentration was
7.5 mg/g. The batch was concentrated to remove acetonitrile on a
Rotovap (∼20 Torr vacuum and bath temp at 50 °C). Concentration
was judged complete when distillate condensation halts. HPLC indi-
cated that the macrocyclic ester concentration was 13.2 mg/g. NMR
indicated that 3 vol % acetonitrile remained, and the batch weighed 2.83
kg. The solution was then filtered through a thin layer of Solka Floc to
remove DCHA HCl. To the solution was added water (850 g, 30% total
3
charge) over 30 min, and the solution was kept at 22 °C. The batch was
seeded with 0.35 g of crystalline product and aged for 0.5 h for seed bed
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ARTICLE
vacuo at 45 °C for 2 days. The crude macrocylic ester (11.0 g) was
isolated as an off-white solid 75.3% by weight and 95% by area in 65%
overall yield from the Heck step (72% for cyclization step).
Preparation of MK-7009. A 500 mL round-bottom flask equipped
with an overhead stirrer, nitrogen inlet, and thermocouple was charged with
macrocyclic acid 23 in DMF and IPAc (5.37 g in 27 mL of IPAc and 27 mL
of DMF). The solution was set stirring and 8-PTSA (4.39 g, 3.46 mmol)
was added as a solid. Upon dissolution (<10 min), hydroxylpyridine
N-oxide (HOPO, 1.21 g, 3.21 mmol) was added as a solid. The batch
was cooled to 15 °C and DIPEA (2.68 g, 2.08 mmol) was added via addition
Ester Saponification To Produce 23. A 100 mL round-bottom
flask equipped with an overhead stirrer and thermocouple was charged
with a solution of the macrocyclic ester 9 (1.78 g) in THF (9.6 mL) and
cooled to 5 °C. An aqueous solution of lithium hydroxide (1 N, 9.6 mL,
9.6 mmol) was added slowly via addition funnel over 30 min while the
batch temperature was kept below 15 °C. With the same addition funnel,
methanol (1.8 mL) was added over 10 min at 15 °C, after which the
white, heterogeneous mixture was allowed to warm to room tempera-
ture. Upon warming, the solution became homogeneous. After ca.
30 min, the solution turned from light yellow to dark brown. The
reaction, sampled at this time, was judged complete by HPLC analysis
(>99.9A% conversion). Concentration of the acid 23 was 85.2 mg/g .
The batch was cooled to 5 °C and 1 N HCl (11.2 mL) added. The batch
was warmed to 20 °C and diluted with IPAc (18 mL, 10 vol). After
agitating for 15 min, the layers were separated, and the organic layer was
collected (HPLC assay showed 1.7 g macrocyclic acid, 98% assay yield).
The IPAc solution was treated with 0.69 g of Darco KB-G (40 wt %) at
20 °C for 10 min, and the solution was filtered through Solka Floc. The
IPAc solution was concentrated under reduced pressure, with the
temperature was kept below 25 °C, to 10 mL and flushed with 10 mL
of IPAc. The solution was diluted with DMF (8 mL) and the concentra-
tion was continued until the final batch volume was 8 mL. The batch was
diluted with DMF (2 mL) and IPAc (8 mL) to approximately 91.7 mg/g
23 concentration.
funnel while the temperature was maintained below 20 °C. Solid EDC HCl
3
(2.65 g, 1.38 mmol, 1.4 equiv) was added. After 3 h, the reaction was judged
complete by HPLC (>99.8 A% conversion, 94% assay yield, 7.01 g). The
batch was transferred to a 500 mL separation funnel and diluted with IPAc
(100 mL) and water (100 mL). After the first cut, the organic layer was
washed with 50 mL of 1 M HCl, water (50 mL), and brine (50 mL). The
IPAc solution was concentrated and flushed with ethanol (50 mL) until
there was 2.5 mol % IPAc in ethanol, as judged by 1H NMR spectroscopy.
HPLC assay indicated 6.86 g of MK-7009 (92% yield).
MK-7009 Neutral Isolation. MK-7009 (11 g by assay) in 50 mL
of wet IPAc (0.37% water) was added simultaneously with 63 mL of
heptane to a seed bed of MK-7009 (1.2 g) in 24 mL of 57/43 v/v
heptane/IPAc at 50 °C over a 12 h period. High seed loading was used to
ensure high-quality crystal growth and consistent, high purity of the
isolated product. An additional 31 mL of heptane ws added over 3 h,
bringing the solvent composition to 65/35 (v/v) heptane/IPAc. The
batch is then cooled over 3 h back to 20 °C. The batch was filtered and
the filter cake washed with 22 mL of 65/35 (v/v) heptane/IPAc and then
washed twice more with 22 mL of heptane each. The recovered wet cake
was a heptane solvate. The cake was dried under vacuum with nitrogen
sweep at 70 °C to generate pure MK-7009.3 The typical yield is 90ꢀ95%. Mp
175ꢀ177 °C; 1H NMR (500 MHz, CDCl3) δ 9.99 (s, 1H), 7.21 (t, J =
7.5 Hz, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.85 (s,
1H), 5.55 (d, J = 9.7 Hz, 1H), 5.32 (t, J = 3.4 Hz, 1H), 4.74 (d, J = 14.6
Hz, 1H), 4.68 (d, J = 14.6 Hz, 1H), 4.54 (d, J = 14.7 Hz, 1H), 4.47 (d, J =
14.7 Hz, 1H), 4.45ꢀ4.43 (om, 2H), 4.35 (dd, J = 10.5, 6.8 Hz, 1H), 4.16
(d, J = 11.7 Hz, 1H), 3.85 (dd, J = 11.7, 3.4 Hz, 1H), 3.26 (d, J = 10.7 Hz,
1H), 2.91 (tt, J = 8.0, 4.8 Hz, 1H), 2.61 (dd, J = 14.2, 6.8 Hz, 1H), 2.51
(m, 1H), 2.40 (m, 1H), 2.30 (ddd, J = 14.2, 10.7, 3.4 Hz, 1H), 1.69 (dd,
J = 8.3, 5.4 Hz, 1H), 1.63 (m, 1H), 1.55 (m, 1H), 1.48 (m, 2H), 1.41 (m,
1H), 1.37, m (1H), 1.34ꢀ1.24 (om, 5H), 1.15 (m, 1H), 1.05 (s, 9H),
1.03 (m, 2H), 0.96 (s, 3H), 0.95 (t, J = 7.4 Hz, 3H), 0.79 (s, 3H); 13C
NMR (125 MHz, CDCl3) δ 172.7, 172.0, 169.6, 156.6, 153.7, 137.8,
136.3, 135.0, 128.3, 127.9, 120.4, 74.4, 73.0, 59.6, 59.3, 54.8, 52.7, 51.1,
Preparation of Side Chain 8-PTSA Salt.45. Hydrogenation.
Compound 24 (12.7 g, 364 mmol, >99% ee) was charged to a
hydrogenation vessel, followed by 1.3 g of 5% ruthenium on carbon
catalyst. The hydrogenation vessel was then charged with 60 g of
methanol (KF < 400 ppm) and pressured to 50 psi with hydrogen,
and the batch was aged for 5.5 h at 20ꢀ25 °C. The end of reaction
sample revealed that the desired conversion was reached (<2% starting
material). The batch was then filtered through Solk Floc and the cake
washed with more methanol. The combined solution was vacuum
concentrated to about 80 mL and solvent switched to 2-propanol with
addition of 160 g of 2-propanol. The final volume of product solution
was 150 mL. This solution was used in the next step. The assay yield of
25 is ca. 90%. Chiral HPLC showed >99% ee.
Boc Removal To Produce Side Chain 8-PTSA. To the product
solution in 2-propanol from the hydrogenation step was added 8.8 g
of p-toluenesulfonic acid monohydrate (PTSA, 463 mmol), with 15 g of
2-propanol flush. The batch was then heated to 63ꢀ67 °C over ∼1 h to
control off gassing and aged for 6 h at 63ꢀ67 °C for complete reaction.
The batch was then cooled to 45 °C and 37.1 g of n-heptane was charged
while the batch was maintained at 42ꢀ46 °C. A slurry formed. The batch
was cooled to 15ꢀ25 °C, aged for 1 h, and then filtered, and the filtrate
was collected (159.8 g) before washing the cake with 30 g of n-heptane at
15ꢀ25 °C. The cake wash (54.1 g) was collected into a separate
container, and the wet cake was blown dry with nitrogen at
15ꢀ25 °C. The dry cake of the title compound was collected as a white
crystalline solid (11.2 g, 75% yield for the hydrogenation and Boc
removal). The solid has <0.5 wt % residue solvent, 99.8% ee, 99.3 wt %,
and 99.9 A% by HPLC. Mp 218ꢀ220 °C; 1H NMR (400 MHz,
CD3OD) δ 7.71 (m, 2H), 7.24 (m, 2H), 3.06 (m, 1H), 2.37 (s, 3H),
1.84 (t, J = 7.5 Hz, 1H), 1.70ꢀ1.58 (om, 2H), 1.57ꢀ1.48 (om, 2H), 1.31
(m, 1H), 1.23 (m, 1H), 1.17ꢀ1.07 (om, 2H), 1.04 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, CD3OD) δ 169.6, 143.6, 141.9, 130.0, 127.1, 41.7,
32.3, 30.1, 21.6, 21.4, 17.6, 13.5, 6.8, 6.7; [α]25859 = ꢀ54.15 (1 g/100 mL
MeOH); EIMS M + 1 = 233, calcd for C9H16NzO3S = 232. Anal. Calcd
for for C16H24N2O6S2: C, 47.51; H, 5.98; N, 6.93; S, 15.85. Found C,
47.36; H, 5.66; N, 6.83; S, 15.53.
39.9, 37.1, 36.7, 35.8, 35.1, 34.4, 33.8, 31.5, 30.9, 26.6, 25.2, 24.0, 23.6,
25
589
23.1, 20.0, 13.8, 6.4, 6.1; [α] = ꢀ40.84 (1.4 g/100 mL MeOH). Anal.
Calcd for C38H55N5O9S C, 60.22; H, 7.31; N, 9.24; S, 4.23. Found C,
59.82; H, 6.92; N, 9.16; S, 4.29.
MK-7009 K Salt Formation. To 75 mL round-bottom flask was
added MK-7009 solution (2.037 g in 18 g total) in ethanol. The batch
was heated to 50 °C and potassium hydroxide solution in ethanolꢀ
water was added slowly (893 mg, 17.8 wt % aqueous KOH mixed with
6.7 mL of ethanol, 1.05 equiv). Seed was added after 1 mL of the KOH
solution was charged. The rest of the KOH solution was added over 30
min after aging the seed bed for 20 min. The addition funnel was
rinsed with 2 mL of ethanol. The batch was stirred at 50 °C for 1 h and
then cooled to rt over 3 h and stirred overnight. The solid was
collected by filtration and washed with 3 mL of ethanol and dried on
the filter via suction under 26ꢀ29% relative humidity (>15% RH
required to produce the right crystal form). Alternatively, the drying
process can be carried out at 50 °C with humid nitrogen sweep
(humidity 15ꢀ50% relative humidity). MK-7009 potassium salt was
obtained as off white crystalline solid (2.16 g). HPLC analysis showed
99 A%, 93% wt K salt (91% yield after correcting for purity and the
seed, also present was water 4.3%, ethanol 0.8%, dimer 0.4%).3 1
H
NMR (600 MHz, DMSO-d6) (MK-7009 K salt exists as an approx-
imate 87:13 ratio of amide bond rotamers, only data for the major
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ARTICLE
rotamer are reported) δ 7.94 (s, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.15 (d,
J = 7.6 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H), 6.90 (d, J = 9.4 Hz, 1H), 5.15
(t, J = 3.8 Hz, 1H), 4.61 (s, (2H), 4.48 (s, 2H), 4.32 (dd, J = 9.4, 7.9 Hz,
1H), 4.27 (d, J = 9.4 Hz, 1H), 4.22 (d, J = 10.6 Hz, 1H), 3.93 (d, J =
12.1 Hz, 1H), 3.77 (dd, J = 12.1, 3.4 Hz, 1H), 3.21 (d,
J = 10.6 Hz, 1H), 2.72 (m, 1H), 2.51ꢀ2.36 (om, 3H), 2.15 (m,
1H), 1.58ꢀ1.47 (om, 2H), 1.44 (m, 2H), 1.27ꢀ1.21 (om, 3H), 1.19
(dd, J = 7.6, 4.2 Hz, 1H), 1.11 (m, 1H), 1.07 (m, 1H), 0.95 (s, 9H),
0.93 (s, 3H), 0.88 (t, J = 7.6 Hz, 3H), 0.80 (dd, J = 9.4, 4.2 Hz, 1H),
0.75 (m, 1H), 0.60 (m, 1H), 0.73 (s, 3H); 13C NMR (150 MHz,
DMSO-d6) δ 174.8, 170.4, 169.4, 156.2, 153.3, 137.0, 136.2, 135.0,
127.8, 127.4, 120.3, 74.4, 71.1, 58.9, 58.4, 54.0, 52.1, 50.3,40.8,
Wan, B.-L.; Olsen, D. B.; Vacca, J. P. J. Am. Chem. Soc. 2008, 130,
4607–4609.
(7) Zhan 1B catalyst from Zanan Pharma: 1,3-bis(2,4,6-trimethyl-
phenyl)-4,5-dihydroimidazol-2-ylidene[2-(isopropoxy)-5-(N,N-dimethyl-
aminosulfonyl)phenyl]methyleneruthenium(II) dichloride. Neolyst M1
catalyst from Umicore: bis(triicyclohexylphosphine)[(phenylthio)methyl-
ene]ruthenium(II) dichloride
(8) Additional details can be found in the Supporting Information.
(9) The process chemistry group of Boehringer-Ingelheim have
developed routes to macrocycles by employing the ring closing metath-
esis reaction, see refs 10ꢀ12.
(10) Farina, V.; Shu, C.; Zeng, X.; Wei, X.; Han, Z.; Yee, N. K.;
Senanayake, C. H. Org. Process Res. Dev. 2009, 13, 250–254.
(11) Shu, C.; Zeng, X.; Hao, M.-H.; Wei, X.; Yee, N. K.; Busacca,
C. A.; Han, Z.; Farina, V.; Senanayake, C. H. Org. Lett. 2008, 10,
1303–1306.
36.6, 35.8, 35.1, 33.9, 32.6, 30.4, 30.2, 29.3, 26.4, 24.9, 23.2, 23.0,
25
365
19.8, 19.2, 13.8, 4.0, 3.9; [α]
MeOH/water).
= ꢀ199.9 (1.1 g/100 mL, 95/5
(12) Yee, N. K.; Farina, V.; Houpis, I. N.; Haddad, N.; Frutos, R. P.;
Gallou, F.; Wang, X.-J.; Wei, X.; Simpson, R. D.; Feng, X. J. Org. Chem.
2006, 71, 7133–7145.
’ ASSOCIATED CONTENT
(13) For examples of Heck macrocyclizations, see refs 14ꢀ17.
(14) Batenburg-Nguyen, U.; Ung, A. T.; Pyne, S. G. Tetrahedron
2008, 65, 318–327.
(15) Carr, J. L.; Offermann, D. A.; Holdom, M. D.; Dusart, P.; White,
A. J. P.; Beavil, A. J.; Leatherbarrow, R. J.; Lindell, S. D.; Sutton, B. J.;
Spivey, A. C. Chem. Commun. 2010, 46, 1824–1826.
(16) Yokoyama, H.; Satoh, T.; Furuhata, T.; Miyazawa, M.; Hirai, Y.
Synlett 2006, 16, 2649–2651.
S
Supporting Information. HPLC analysis conditions for
b
compounds 5, 8, 9, 15, 16, 18, 19, 22, 23, and MK-7009; chiral
assay methods for compounds 5, 8, and 15; NMR spectra files for
compounds 5-DCHA, 8-PTSA, 9, 15, 18-PTSA, 19, and MK-
7009. This material is available free of charge via the Internet at
http://pubs.acs.org/.
(17) Menche, D.; Hassfeld, J.; Li, J.; Mayer, K.; Rudolph, S. J. Org.
Chem. 2009, 74, 7220–7229.
’ AUTHOR INFORMATION
(18) For examples of Suzuki macrocyclizations, see refs 19ꢀ21.
(19) Gibson, S. E.; Lecci, C.; White, A. J. P. Synlett 2006, 18,
2929–2934.
Corresponding Authors
*E-mail: zhiguo_song@merck.com, david_tellers@merck.com.
(20) Tortosa, M.; Yakelis, N. A.; Roush, W. R. J. Org. Chem. 2008,
73, 9657–9667.
’ ACKNOWLEDGMENT
(21) White, J. D.; Tiller, T.; Ohba, Y.; Porter, W. J.; Jackson, R. W.;
Wang, S.; Hanselmann, R. Chem. Commun. 1998, 1, 79–80.
(22) For a comparison of Heck, Suzuki, and Sonogashira in an
acyclic system, see: Brown Ripin, D. H.; Bourassa, D. E.; Brandt, T.;
Castaldi, M. J.; Frost, H. N.; Hawkins, J.; Johnson, P. J.; Massett, S. S.;
Neumann, K.; Phillips, J.; Raggon, J. W.; Rose, P. R.; Rutherford, J. L.;
Sitter, B. A.; Stewart, M., III; Vetelino, M. G.; Wei, L. Org. Process Res.
Dev. 2005, 9, 440–450.
(23) For examples of macrolactamization reactions, see refs 24ꢀ28.
(24) Ohtani, T.; Tsukamoto, S.; Kanda, H.; Misawa, K.; Urakawa, Y.;
Fujimaki, T.; Imoto, M.; Takahashi, Y.; Takahashi, D.; Toshima, K. Org.
Lett. 2010, 12, 5068–5071.
The author would like to thank our colleagues who gave
their support of this project: Nigel Liverton, Michael Rudd,
John Butcher, John McCauley, Charles McIntyre, Joe Ro-
mano, Birgit Kosjek, Yaling Wang, Scott Thomas, Margaret
Figus, Jake Janey, Laura Artino, Osama Sudah, Vincent
Capoddano, Richard Desmond, Brian Bishop, Adrian Good-
year, Mike Ashwood, Gary Javadi, Lisa DiMichele, and
J. Chris McWilliams.
’ REFERENCES
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(29) This ratio was determined after the hydrogenation reaction
where the ratio of saturated species was quantified by HPLC. Attempts
to identify the exact structure of the various olefin isomers by NMR were
unsuccessful. The slow degradation of these olefins in air also hampered
the isolation attempts.
(30) It appears that the Heck reaction between unactivated,
alkyl-substituted terminal olefins and aromatic halides to produce
exclusively the linear product is an unsolved problem. One potential
solution is discussed in the following reference: Littke, A. F.; Fu,
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ARTICLE
(34) For a review on reagents for amide bond formation, see:
Han, S.-Y.; Kim, Y. A. Tetrahedron 2004, 60, 2447–2467. The
reaction screen using reagents listed in Figure 2 was carried out in
DMF at room temperature at about 5 mg/mL substrate concentra-
tion. The starting material was a mixture of 22 and 22-exo in 88/13
ratio. The yields listed are solution assay yields of the desired
cyclization isomer 9 from 22 only, corrected for the starting
material purity.
(45) The side chain was prepared from known compound 24
(ref 38) according to the scheme below.
(35) Yield adjusted for purity of the starting material 16.
(36) For an example of HOPO use in amide bond formations, see:
Shi, Y.-J.; Cameron, M.; Dolling, U. H.; Lieberman, D. R.; Lynch, J. E.;
Reamer, R. A.; Robbins, M. A.; Volante, R. P.; Reider, P. J. Synlett
2003, 647–650.
(37) While not examined in great detail, the rate of macrocycliza-
tion of the undesired exo isomers 16-exo did not vary substantially
from 16.
(38) Beaulieu, P. L.; Gillard, J.; Bailey, M. D.; Boucher, C.; Duceppe,
J. S.; Simoneau, B.; Wang, X.-J.; Zhang, L.; Grozinger, K.; Houpis, I.;
Farina, V.; Heimroth, H.; Krueger, T.; Schnaubelt, J. J. Org. Chem. 2005,
70, 5869.
(39) For an asymmetric synthesis, see: Belyk, K. M.; Bangping, X.;
Bulger, P. G.; Leonard, W. R., Jr.; Balsells, J.; Yin, J.; Chen, C.-Y. Org.
Process Res. Dev. 2010, 14, 692–700.
(40) The Suzuki sp2ꢀsp3 was also examined. Preparation of crude
alkyl borane was effected by hydroboration of linker 5. This reaction was
complicated by formation of a variety of unidentified species. Subse-
quent catalyst screening of the coupling was performed and product was
formed. Route development was halted on this transformation as well
given the complex mixture generated from the hydroboration. Also, for
an example of the Suzuki reaction in natural product synthesis, see refs
41, 42.
(41) Chemler, S. R.; Trauner, D.; Danishefsky, S. J. Angew. Chem. Int.
Ed. 2001, 40, 4544–4568.
(42) Nicolau, K. C.; Bulger, P. G.; Sarlah, D. Angew. Chem. Int. Ed.
2005, 44, 4442–4489.
(43) Soheili, A.; Albaneze-Walker, J.; Murry, J. A.; Dormer, P. G.;
Hughes, D. L. Org. Lett. 2003, 5, 4191–4194.
(44) In contrast to the ligandless Heck reaction, the Sonagashira
reaction mixture could not be taken directly into the hydrogenation
reaction as relatively poor conversions were obtained at high Pd/C
loadings. We attribute this to catalyst poisoning by residual phosphine
catalyst. As a result, aqueous workup followed by activated carbon
treatment was required.
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