Phase Separation Macrocyclization in a Complex Pharmaceutical Setting: Application toward the Synthesis of Vaniprevir

Article abstract of DOI:10.1021/acs.joc.7b01308

A phase separation/continuous flow strategy employing an oxidative Glaser-Hay coupling of alkynes has been applied toward the synthesis of the macrocyclic core of complex pharmaceutical vaniprevir. The phase separation/continuous flow strategy afforded similar yields at 100-500 times the concentration and at shorter reaction times than common slow addition/high dilution techniques. In addition, dendritic PEG cosolvents were employed in the phase separation strategy for the first time and shown to allow productive macrocyclization at concentrations up to 200 mM.

Full text of DOI:10.1021/acs.joc.7b01308

Article
pubs.acs.org/joc
Phase Separation Macrocyclization in a Complex Pharmaceutical
Setting: Application toward the Synthesis of Vaniprevir
́
Eric Godin, Anne-Catherine Bed
́
̈
ard, Michael Raymond, and Shawn K. Collins*
Dep
Queb
́
artement de Chimie, Centre for Green Chemistry and Catalysis, Universite
ec, H3C 3J7 Canada
́ ́ ́
de Montreal, CP 6128 Station Downtown, Montreal,
́
*
S Supporting Information
ABSTRACT: A phase separation/continuous flow strategy
employing an oxidative Glaser−Hay coupling of alkynes has
been applied toward the synthesis of the macrocyclic core of
complex pharmaceutical vaniprevir. The phase separation/
continuous flow strategy afforded similar yields at 100−500
times the concentration and at shorter reaction times than
common slow addition/high dilution techniques. In addition,
dendritic PEG cosolvents were employed in the phase
separation strategy for the first time and shown to allow productive macrocyclization at concentrations up to 200 mM.
INTRODUCTION
ability of poly(ethylene)glycol (PEG) cosolvents to form
lipophilic aggregates in solution with an accompanying
hydrophilic solvent, whereby the aggregates preferentially
solubilize organic substrates. Slow diffusion of a linear precursor
out of a PEG aggregate into the MeOH cosolvent and
subsequent cyclization is believed to mimic slow addition
conditions. Through judicious control of the ratio of
PEG:MeOH, the diffusion of the linear precursor can be
controlled to optimize the preference for macrocyclization
versus oligomerization. In general, higher ratios of PEG:MeOH
■
The macrocycle chemotype has made significant contributions
to drug discovery due to unique properties that can allow for
scanning of new chemical space. Consequently, the interest in
macrocycles in drug discovery has experienced significant
growth in the past decade. Terrett and co-workers have
highlighted that current macrocyclic drugs are almost
exclusively derived from naturally occurring macrocycles, with
1
2
3
modifications typically occurring only at specific positions.
Synthetic macrocycles have come to represent a growing class
of drug candidates, yet their diversity remains limited by the
number of robust synthetic techniques amenable to library
(
2:1 → 8:1) allow for macrocyclization processes to be
conducted at much higher concentrations all the while affording
higher yields.
4
generation. One of the major obstacles to any macrocycle
Consequently, the phase separation strategy represents an
effort to apply the principles of sustainability to macro-
synthesis involves surmounting the difficult ring closing event.
Indeed, Terrett and co-workers also stated,“The only residual
difficulty in macrocycle synthesis is finding conditions that
allow good yields of cyclized materials from acyclic precursors....
For single compounds, this is usually achieved by resorting to
the use of large reaction volumes and low reactant
concentrations; conditions that were devised many years
7
cyclization chemistry. The phase separation strategy has
recently been exploited by Itami and co-workers for the
synthesis of thiophene-based macrocycles for materials science
8
applications, however no demonstrations of the utility of the
phase separation strategy toward medicinal chemistry have
been reported. Herein we report on the application of a phase
separation strategy employing the rarely exploited Glaser−Hay
coupling for the synthesis of a complex pharmaceutical target.
3
ago.” A representative example of the difficulty of optimizing
a macrocyclization protocol was shown in the synthesis of the
macrocyclic core of vaniprevir, a macrocyclic hepatitis C virus
(
HCV) NS3/4A protease inhibitor developed by Merck & Co.
RESULTS AND DISCUSSION
■
which was recently approved for treating hepatitis C in 2014 in
Japan (Figure 1). A macrolactamization protocol provided
5
Exploration of Different PEG Cosolvents for Macro-
cyclization via Phase Separation. The phase separation
strategy relies upon the ability of PEG cosolvents to form
lipophilic aggregates in MeOH solutions. Aggregation can be
confirmed via surface tension measurements. It has been
shown that the structural characteristics of the PEG cosolvent
encouraging yields, but suffered from the use of stoichiometric
reagents, while many catalytic cross-coupling routes provided
undesirable yields. An olefin metathesis route proved optimal
and could be conducted with low catalyst loading (0.2 mol %)
at a concentration of 120 mM, although slow addition
techniques were employed. Our group has reported a phase
separation strategy as a novel macrocyclization strategy
permitting catalytic transformations at relatively high concen-
9
10
can affect both its aggregation behavior and catalysis. For
Received: May 26, 2017
6
trations. The ability to control dilution effects rests upon the
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.7b01308
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13
media, properties which would also be highly beneficial for
developing a phase separation macrocyclization process. Two
StarPEGs were chosen for study with average molecular
−1
weights of 450 and 1014 g·mol . The StarPEGs were selected
so that their behavior could be compared to linear PEGs that
had similar average molecular weights (400 and 1450
respectively). Surface tension measurements demonstrated
that StarPEGs exhibit aggregation behavior in MeOH similar
to the other PEG cosolvents: aggregation occurred in ratios of
1
4
1
:1 up to 8:1 StarPEG:MeOH. Before exploring the
macrocyclization of the vaniprevir precursor, preliminary
studies were performed in batch to compare the ability of the
selected PEG polymers to control dilution effects at
15
concentrations of 24, 100, and 200 mM (Figure 3). The
macrocyclization of model bis-alkyne 2 was chosen for initial
investigations as its aliphatic structure is relatively free of
structural biases which could influence its ability to undergo
macrocyclization. Control reactions in either pure MeOH or
pure PEG solvent are important for establishing whether phase
separation is proving beneficial to the macrocyclization process.
Control macrocyclizations (2 → 3, Figure 3, top) performed in
pure MeOH showed low yields of macrocycle 3 at 24 mM
(
22%), and only traces could be observed at either 100 or 200
mM. Control reactions performed in 100% PEG400, StarPEG450
or PEG1450, at 24 mM, all afforded yields of macrocycle 3 below
%. In sum, the control reactions all establish that phase
,
5
separation provided by the mixtures of PEG cosolvent:MeOH
is beneficial. An exception was the macrocyclization in
StarPEG1014, where the yield of 3 was 43%, implicating that,
for phase separation to be successful in the dendritic PEG
system, yields of 3 would have to exceed 43%. Also of note:
macrocyclization in 100% PEG at 100 or 200 mM was not
possible due to limited solubility of the catalysts and/or bis-
alkyne 2. The first macrocyclizations in PEG polymer:MeOH
mixtures were performed at 24 mM at a ratio of 2:1 PEG
polymer:MeOH, which had previously been shown to provide
high yields of products (Figure 3, bottom). When macro-
cyclization was carried out in previously reported 2:1
PEG400:MeOH mixture, the yield of 3 was 77%, and a similar
Figure 1. Macrocyclization strategies toward vaniprevir (ring size
indicated in red).
example, hydroxyl-terminated PEGs are important for inducing
aggregation in MeOH and the ability to observe high
macrocyclization efficiency. More lipophilic solvents, such as
longer chain PEGs, or poly(propylene)glycol (PPG) solvents,
allow for catalysis at lower catalyst loadings.
The macrocyclization investigations via Glaser−Hay coupling
were to conducted using reaction conditions previously
yield of 78% was obtained for the analogous 2:1 StarPEG450
/
6
developed by our group, which exploit a Ni-based cocatalyst
MeOH. When using the linear 2:1 PEG1450:MeOH mixture, the
yield of 3 was 68%, but increased dramatically in 2:1
StarPEG1014:MeOH to afford a 91% yield of macrocycle 3.
Next, macrocyclization was performed at concentrations of
11
to accelerate the rate of oxidative coupling, under microwave
6
heating. Based upon previous reports, PEG
00
and PEG1450
4
were selected to be evaluated in the macrocyclization to form
the core of vaniprevir (Figure 2). In addition, branched
dendritic or “StarPEG” polymers were also explored for the first
time in macrocyclizations via phase separation (Figure 2).
Dendritic PEGs exhibit low cytotoxicities and have found use in
1
00 and 200 mM with 8 h reaction times, as the preliminary
results demonstrated that macrocyclizations in the StarPEG
solvents often proceeded more slowly. When macrocyclization
was carried out in previously reported 2:1 PEG400:MeOH
mixture, yields of 3 dropped from 77% (24 mM) to 22% when
the concentration was pushed to 200 mM. Reactions performed
in the analogous 2:1 StarPEG450/MeOH mixture provided
slightly higher yields, however at the target 200 mM
concentration, only 32% of 3 could be isolated. Further
improvements were observed when PEG polymers with higher
average molecular weights were used as cosolvents. In the linear
12
drug delivery applications. In addition, StarPEGs are known
to have an enhanced ability to encapsulate hydrophilic and
hydrophobic guests as well as sustained release in aqueous
2
:1 PEG1450:MeOH mixture, the initial yield of 3 at 24 mM was
only 68%, however the yield dropped much less appreciably
when the macrocyclization was performed at 200 mM (40% of
3
). Finally, the best PEG polymer mixture surveyed was the 2:1
StarPEG1014:MeOH, where at 24 mM a 91% yield of
macrocycle 3 was isolated and at 200 mM a 64% yield was
obtained.
Figure 2. PEG cosolvents for study in a “phase separation”
macrocyclization process.
B
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Scheme 1. Synthesis of Isoindoline 6
Scheme 2. Synthesis of an Acyclic Diyne Precursor 10
coupling with the isoindoline 6 afforded the desired macro-
cyclization precursor 10 in 91% yield.
Exploration of Different PEG Cosolvents for Macro-
cyclization toward the Core of Vaniprevir. Following the
survey of different PEG:MeOH solvent mixtures for the
macrocyclization of the model bis-alkyne 2, a similar survey
was undertaken for the cyclization of bis-alkyne 10 toward the
macrocyclic core of vaniprevir 11. With the goal of maximizing
the efficiency of the process, it was decided to explore
17
continuous flow methods for macrocyclization (10 → 11).
It had already been demonstrated that the phase separation
strategy for macrocyclization was amenable to continuous flow
and that the associated efficient energy and mass transfer
18
resulted in improved reaction times and yields. As such, the
macrocyclization of bis-alkyne 10 was performed by injecting
the entire reaction mixture into a flow system consisting of two
stainless steel reactors in series heated to 120 °C. As the
Glaser−Hay reaction is an oxidative process, the first of the
reactors was a tube-in-tube reactor equipped to saturate the
Figure 3. Effect of solvent and concentration on macrocyclization (2
→
3). Top: In pure MeOH, linear PEG, and StarPEG solvents.
Bottom: Solvent mixtures of linear PEGs and StarPEGs in MeOH.
Macrocyclizations were performed at 24 mM (blue, 6 h), 100 mM
a
(
green, 8 h), and 200 mM (purple, 8 h). All entries represent isolated
yields following silica gel chromatography. Ring size indicated in red.
19
reaction mixture with oxygen. It is important to note that the
bis-alkyne 10 is structurally much more complex than the
model bis-alkyne 2. It is difficult to predict a priori whether the
additional functionalization in 10 creates a bias toward
productive macrocyclization, or undesired dimerization or
Synthesis of the Bis-Alkynyl Macrocyclization Pre-
cursor. The exploration of using a phase separation strategy
employing a Glaser−Hay coupling began with the synthesis of
the required alkynyl building block isoindoline 6 (Scheme 1).
The isoindoline 6 could be prepared from the commercially
available bromide 4 via Boc protection, Sonogashira coupling,
and deprotection of the trimethylsilyl and Boc protecting
groups. The remaining peptidic portion of the macrocyclic
20
oligomerization. The first macrocyclizations (10 → 11)
investigated were control reactions performed in 100% MeOH
at three different concentrations (Figure 4, top). The
macrocyclization at 24 mM afforded low yields (29% of 11),
and slightly lower yields were obtained at 50 mM (22%) and
21
100 mM (24%). Next, a second set of control reactions were
performed whereby macrocyclization was conducted in 100%
PEG at 24 mM. For macrocyclization in PEG400, the isolated
yield of macrocycle 11 was low (13%), but for the analogous
dendritic StarPEG450, the yield was again much greater (43% of
11), demonstrating the increased reactivity observed with more
16
precursor 10 was prepared from the known alkynyl alcohol 7,
which was converted to the carbamate 8 in 92% yield using
CDI and tert-leucine (Scheme 2). The following amide linkage
was formed via coupling of the methyl ester of hydroxyproline
to afford the dipeptide 9 in 92% yield. Lastly, a CDI-mediated
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Next, the macrocyclization was investigated in the
PEG:MeOH mixtures (Figure 4, bottom). Given the higher
yields observed with StarPEG1014 obtained in the macro-
cyclization of the simple model macrocycle 11, the first
cyclization toward the vaniprevir core 11 was investigated in 2:1
14
StarPEG1014:MeOH (24 mM). When 10 was subjected to
macrocyclization in 2:1 StarPEG1014:MeOH, only a 31% yield
of macrocycle 11 was obtained, with complete conversion of
the starting bis-alkyne 10. Reasoning that a better preference
for macrocyclization could be obtained at higher PEG:MeOH
ratios, the macrocyclizations of bis-alkyne 10 were conducted at
4
:1 StarPEG1014:MeOH, which afforded a yield of 55% of 11.
Increasing or decreasing the ratio of PEG:MeOH did not
22
improve the conversion to a significant degree, and a ratio of
:1 PEG polymer:MeOH was used for all further investigations.
4
When comparing StarPEG1014 to the linear analogue PEG1450, it
was found that the yield of macrocycle 11 was slightly lower
(
50%) at 4:1 PEG1450:MeOH. A lower molecular weight
StarPEG450 also gave a comparable yield of 11 (48% at 4:1
StarPEG450:MeOH). However, the best yield of 11 at 24 mM
23
was with a 4:1 mixture of PEG400:MeOH (64%). Next,
attempts were made to increase the concentration of the
reaction to both 50 and 100 mM. After surveying 4:1 mixtures
of PEG400, StarPEG450, and StarPEG1014 with MeOH, the best
yield at either concentration was obtained with 4:1
PEG400:MeOH (38% of macrocycle 11 at 50 mM and 36%
24
of macrocycle 11 at 100 mM).
In an effort to compare the phase separation/continuous flow
protocol to common slow addition/high dilution strategies, the
bis-alkyne 10 was cyclized at low concentration (0.2 mM)
(
Scheme 3). Excess copper/ligand was used to promote
Scheme 3. Comparing Slow Addition/High Dilution and
Phase Separation/Continuous Flow Strategies toward the
a
Vaniprevir Core 11
a
Ring size indicated in red.
macrocyclization in 64% yield of 11 with a reasonable reaction
25
time (48 h). Consequently, the phase separation/continuous
flow strategy provided similar yields at concentrations greater
than 100 times that of slow addition/high dilution strategies. In
addition, the former could promote macrocyclization at 36%
yield at up to 500 times greater concentrations. The
macrocyclic diyne 11 could be hydrogenated to afford the
same macrocyclic intermediate 1 obtained by the Merck
research team (Scheme 4).
Figure 4. Effect of solvent and concentration on macrocyclization (10
→
11). Top: In pure MeOH, linear PEG, and StarPEG solvents.
Bottom: Solvent mixtures of linear PEGs and StarPEGs in MeOH.
Macrocyclizations were performed at 24 mM (blue), 50 mM (gray),
a
1
00 mM (green), and 200 mM (purple). All entries represent isolated
yields following silica gel chromatography. Ring size indicated in red.
lipophilic PEG solvents. In contrast, macrocyclizations were
very inefficient when higher molecular weight PEGs were used.
The linear PEG1450 is a solid at room temperature, making the
reaction under flow conditions difficult to perform, while, for
the dendritic StarPEG1014, a liquid at room temperature, only
trace amounts of the macrocycle 11 could be isolated.
Unfortunately, macrocyclizations at 100 mM could not be
performed due to problematic solubility of substrates and/or
catalysts in the neat PEG solvents.
In summary, the phase separation strategy has been applied
for the first time to macrocyclization of a complex
pharmaceutical target, the macrocyclic core of vaniprevir. The
phase separation strategy demonstrated good functional group
tolerance to the nitrogen-based heterocycles, dipeptides, and
carbamates embedded within the structure of vaniprevir and
provided good yields of the desired macrocyclic core 11 (55−
64%) using either PEG4 :MeOH or newly explored
00
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Article
EtOAc. The organic phases were combined, washed with brine, dried
with Na SO , and concentrated in vacuo. The crude solid (3.3 g, 99%)
Scheme 4. Completing the Synthesis of the Vaniprevir Core
1
a
2
4
was clean. Note that rotamers are formed and can result in complex
1
splitting patterns in the H NMR, or can cause doubling of some peaks in
the 13C NMR spectrum. For clarity, all peaks are reported. H NMR (300
1
MHz, CDCl ) δ = 7.39 (d, J = 8.6 Hz, 1 H), 7.24−7.08 (m, 2 H),
3
13
4
.80−4.57 (m, 4 H), 1.58−1.45 (m, 9 H); C NMR (75 MHz,
CDCl ) δ ppm = 154.3, 139.0, 138.8, 138.0, 137.9, 130.29, 130.25,
3,
1
29.2, 129.1, 121.5, 121.3, 117.6, 117.3, 85.1, 80.0, 79.9, 53.5, 53.4,
5
3.2, 52.9, 28.5, 27.4; HRMS (ESI) m/z calculated for
+
C H BrNO Na [M + Na] , 320.0257; found, 320.0265.
13
16
2
a
tert-Butyl 4-((Trimethylsilyl)ethynyl)isoindoline-2-carboxy-
late (S1). In a sealed tube, 4a (167 mg, 0.56 mmol, 1 equiv) was
Ring size indicated in red.
dissolved in Et N (1.5 mL) and 1,4-dioxane (1.5 mL). The solution
3
was degassed with N for 5 min. Pd(PPh ) Cl (39.3 mg, 0.056 mmol,
2
3
2
2
StarPEG1014:MeOH. In addition, the macrocyclization demon-
strates the utility of the Glaser−Hay oxidative coupling of
alkynes for macrocyclization of pharmaceuticals. Macrocycliza-
tion toward the core of vaniprevir could be conducted at 100
mM using linear or dendritic PEG cosolvents. A “simpler”
macrocyclization on an unbiased substrate could be conducted
at 200 mM in good yields (64% of 3) using the newly explored
dendritic PEGs as cosolvents. As a number of other synthetic
processes rely upon controlling concentration effects, it is
possible that the new dendritic PEG/solvent mixtures could be
used to improve such processes. It is also expected that as the
interest in macrocycles in drug discovery continues to grow, so
will the need for macrocyclization techniques that strive toward
sustainability.
0
.1 equiv) and CuI (5.3 mg, 0.028 mmol, 0.05 equiv) were added, and
the solution was degassed a second time with N2 for 5 min.
Ethynyltrimethylsilane (0.39 mL, 2.8 mmol, 5 equiv) was added, and
the mixture was stirred for 24 h at 100 °C. Upon completion, the
mixture was passed through a pad of Celite and concentrated in vacuo.
Purification by silica gel chromatography (3% EtOAc/hexanes) gave
the desired product as an off-white solid. The product may be
contaminated with residual butadiyne byproduct from homocoupling
of the ethynyltrimethylsilane. As such, yields were calculated following
the purification of product 5 below. Note that rotamers are formed and
26
1
can result in complex splitting patterns in the H NMR, or can cause
13
doubling of some peaks in the C NMR spectrum. For clarity, all peaks are
1
reported. H NMR (400 MHz, CDCl ) δ = 7.37−7.32 (m, 1 H), 7.25−
3
7
.15 (m, 2 H), 4.78−4.66 (m, 4 H), 1.54−1.50 (m, 9 H), 0.29−0.23
m, 9 H); C NMR (75 MHz, CDCl ) δ ppm = 154.4, 140.3, 139.9,
13
(
3
1
1
37.2, 136.9, 130.2, 130.1, 127.4, 127.3, 122.8, 122.5, 118.3, 188.0,
EXPERIMENTAL SECTION
02.0, 101.6, 98.8, 79.7, 79.7, 52.7, 52.4, 52.3, 52.1, 28.5, 28.4, −0.1;
■
+
All reactions that were carried out under anhydrous conditions were
performed under an inert argon or nitrogen atmosphere in glassware
that had previously been dried overnight at 120 °C or had been flame-
dried and cooled under a stream of argon or nitrogen. All chemical
products were obtained from Sigma-Aldrich Chemical Co. or Alfa
Aesar and were reagent quality. Technical solvents were obtained from
VWR International Co. or ACP Chemicals Inc. Anhydrous solvents
HRMS (ESI) m/z calculated for C18
338.1548; found, 338.1547.
H25NO
2
SiNa [M + Na] ,
tert-Butyl 4-Ethynylisoindoline-2-carboxylate (5). To a
27
solution of indoline S1 (1.711 g, 5.43 mmol, 1 equiv) in MeOH
(40 mL) was added K CO (3.75 g, 27.15 mmol, 5 equiv). The
2 3
solution was stirred for 18 h at room temperature. Upon completion
by TLC, EtOAc and H O were added and the layers were separated.
2
(
CH Cl , Et O, THF, DMF, toluene, and n-hexane) were dried and
The aqueous phase was extracted with EtOAc (2×). The organic
2
2
2
deoxygenated using a GlassContour system (Irvine, CA). Bis-alkyne
phases were combined, washed with brine, dried with Na SO , and
2
4
6
a
28
29
2
,
alcohol 7, and hydroxyproline methyl ester were synthesized
concentrated in vacuo. Purification by silica gel chromatography (2.5%
EtOAc/hexanes) gave the terminal alkyne (1.010 g, 84%) as a white
solid. Note that rotamers are formed and can result in complex splitting
according to the literature. Isolated yields reflect the mass obtained
following flash column silica gel chromatography. Organic compounds
30
1
13
were purified using the method reported by W. C. Still and using
silica gel obtained from Silicycle Chemical division (40−63 nm; 230−
patterns in the H NMR, or can cause doubling of some peaks in the
C
1
NMR spectrum. For clarity, all peaks are reported. H NMR (400 MHz,
2
40 mesh). Analytical thin-layer chromatography (TLC) was
CDCl ) δ = 7.40−7.35 (m, 1 H), 7.26−7.18 (m, 2 H), 4.78−4.66 (m,
3
13
performed on glass-backed silica gel 60 coated with a fluorescence
4 H), 3.30−3.24 (m, 1 H), 1.55−1.51 (m, 9 H); C NMR (75 MHz,
indicator (Silicycle Chemical division, 0.25 mm, F254.). Visualization of
CDCl ) δ ppm = 154.4, 154.3, 140.4, 140.2, 137.5, 137.2, 130.8, 130.7,
3
TLC plate was performed by UV (254 nm), KMnO , or p-
127.5, 127.4, 123.2, 122.9, 117.2, 117.0, 81.3, 81.1, 80.7, 80.5, 79.8,
4
anisaldehyde stains. All mixed solvent eluents are reported as v/v
solutions. Concentration refers to removal of volatiles at low pressure
on a rotary evaporator. All reported compounds were homogeneous
79.8, 52.7, 52.4, 52.2, 52.1, 28.5; HRMS (ESI) m/z calculated for
+
C H NO Na [M + Na] , 266.1152; found, 266.1152.
15
17
2
4-Ethynylisoindoline Hydrochloride (6). To a solution of
1
by thin layer chromatography (TLC) and by H NMR. NMR spectra
isoindoline 5 (786 mg, 3.23 mmol, 1 equiv) in MeOH (15 mL) was
added dropwise AcCl (0.86 mL, 16.17 mmol, 5 equiv) at 0 °C. The
resulting mixture was stirred for 18 h (or until completed by TLC) at
were taken in deuterated CDCl using Bruker AV-300 and AV-400
3
instruments unless otherwise noted. Signals due to the solvent served
1
13
as the internal standard (CHCl : δ 7.27 for H, δ 77.0 for C). The
room temperature. Et O was added and a gray precipitate was formed
3
2
1
H NMR chemical shifts and coupling constants were determined
and filtered, washed with Et O (2×), and dried under vacuum to give
2
1
assuming first-order behavior. Multiplicity is indicated by one or more
the desired isoindoline salt as a gray powder (507 mg, 87%). H NMR
of the following: s (singlet), d (doublet), t (triplet), q (quartet), m
(400 MHz, MeOD-d ) δ = 7.52−7.38 (m, 3 H), 4.70−4.66 (m, 4 H),
4
3.93 (s, 1 H); 13C NMR (100 MHz, MeOD-d ) δ ppm = 138.3, 136.3,
(
multiplet), br (broad); the list of coupling constants (J) corresponds
4
to the order of the multiplicity assignment. Mass spectrometric
analyses for nominal masses were performed on a quadrupole analyzer,
while high resolution masses were performed on a TOF analyzer.
tert-Butyl 4-Bromoisoindine-2-carboxylate (4a). To a solution
of 4-bromoisoindoline hydrochloride (4) (2.64 g, 11.2 mmol, 1 equiv)
133.3, 130.6, 124.7, 119.1, 84.5, 80.8, 52.7, 49.8; HRMS (ESI) m/z
+
calculated for C H N [M + H] , 144.0812; found, 144.0808.
10
10
(S)-2-((((2,2-Dimethylbut-3-yn-1-yl)oxy)carbonyl)amino)-3,3-
dimethylbutanoic Acid (8). In a sealed tube, 2,2-dimethylbut-3-yn-
1-ol (7) (1.84 g, 18.7 mmol, 1 equiv) and 1,1′-carbonyldiimidazole
(3.96 g, 24.4 mmol, 1.3 equiv) were dissolved in DMF (20 mL). The
mixture was stirred 2 h at room temperature. Then, L-tert-leucine (3.21
in NaOH 1 M (26 mL) and THF (26 mL) was added Boc O (2.71 g,
2
1
2.4 mmol, 1.1 equiv). The mixture was stirred for 16 h at room
temperature. EtOAc and H O were added to the mixture, and the
g, 24.4 mmol, 1.3 equiv) and Et N (3.74 mL, 26.3 mmol, 1.4 equiv)
2
3
layers were separated. The aqueous phase was extracted 2× with
were added, and the resulting mixture was warmed to 90 °C and
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stirred for 16 h. Then reaction was then cooled back to room
temperature, and MTBE and NaOH 0.5 M were added. The layers
were separated, and the organic layer was discarded. MTBE was added
to the aqueous phase, and the pH was adjusted to 1 using HCl 6 M.
The layers were separated again. The organic phase was washed with
Macrocycle (11). Slow Addition Procedure. To a 1 L triple-
neck round-bottom flask equipped with a stirring bar and a condenser,
CuCl (119 mg, 1.2 mmol, 12 equiv) and TMEDA (0.3 mL, 2.0 mmol,
20 equiv) were added to PhMe (405 mL). The mixture was stirred and
warmed at 110 °C. To the mixture, a solution of bis-alkyne 10 (55.2
mg, 0.1 mmol, 1 equiv) in PhMe (50 mL) was slowly added over 24 h
brine, dried with MgSO , and concentrated in vacuo. The crude
4
25
(0.035 mL/min) with a syringe pump. The mixture was stirred and
carboxylic acid (4.4 g, 92%) was obtained as a sticky semisolid. [α]D
=
heated for an additional 24 h. The reaction was then cooled down to
room temperature and concentrated under reduced pressure. Flash
chromatography was performed (40 → 60% EtOAc in hexanes) to
afford the desired product as a white solid (35 mg, 64%). Mp = 136.4
4
.0 (c = 0.0030, MeOH). Note that rotamers are formed and can result in
1
complex splitting patterns in the H NMR, or can cause doubling of some
peaks in the 1 C NMR spectrum. For clarity, all peaks are reported. H
3
1
NMR (300 MHz, CDCl ) δ = 11.22 (br s, 1 H), 6.50 (d, J = 7.8 Hz,
3
C; [α]2 = −32.0 (c = 0.0020, MeOH); H NMR (500 MHz, CDCl )
5
1
°
0
3
.3 H), 5.43 (d, J = 9.5 Hz, 0.7 H), 4.19 (d, J = 9.5 Hz, 0.7 H), 4.05−
D
3
.90 (m, 2.3 H), 2.11 (s, 1 H), 1.22 (s, 6 H), 1.01 (s, 9 H); 13C NMR
δ = 7.30−7.26 (m, 1H), 7.25−7.21 (m, 2H), 5.71 (d, J = 9.1 Hz, 1H),
5
4
.30−5.26 (m, 1H), 4.83 (d, J = 10.5 Hz, 1H), 4.76−4.73 (m, 2H),
.64−4.59 (m,2H), 4.59−4.56 (m, 1H), 4.43 (d, J = 9.1 Hz, 1H), 4.07
(
75 MHz, CDCl ) δ ppm = 176.2, 157.0, 156.3, 88.7, 88.1, 72.8, 72.0,
3
6
9.1, 68.9, 63.2, 62.1, 34.6, 33.9, 31.7, 26.4, 25.6; HRMS (ESI) m/z
+
(dd, J = 11.8, 1.6 Hz, 1H), 3.85 (dd, J = 11.7, 3.6 Hz, 1H), 3.76 (s,
calculated for C H NO Na [M + Na] , 278.1365; found, 278.1363.
13
21
4
3
1
H), 3.29 (d, J = 10.5 Hz, 1H), 2.75−2.69 (m, 1H), 2.16−2.09 (m,
Methyl (2S,4R)-1-((S)-2-((((2,2-Dimethylbut-3-yn-1-yl)oxy)-
carbonyl)amino)-3,3-dimethylbutanoyl)-4-hydroxypyrroli-
dine-2-carboxylate (9). Carboxylic acid (8) (1.03 g, 3.48 mmol, 1
equiv) was dissolved in MeCN (3 mL) at room temperature. trans-4-
Hydroxy-L-proline methyl ester hydrochloride (695 mg, 3.83 mmol,
H), 1.34 (s, 3H), 1.23 (s, 3H), 1.06 (s, 9H); 13C{ H} NMR (125
1
MHz, CDCl ) δ ppm = 172.0, 170.4, 155.3, 153.3, 144.4, 136.2, 129.4,
3
1
5
27.9, 123.1, 117.1, 89.6, 78.7, 74.3, 72.2, 71.4, 64.7, 59.4, 57.7, 53.8,
2.8, 52.3, 52.1, 37.2, 35.6, 33.1, 26.3, 25.4, 25.3; HRMS (ESI) m/z
+
calculated for C H N O [M + H] , 550.2565; found, 550.2549.
1
.1 equiv) and pyridine (0.42 mL, 5.22 mmol, 1.5 equiv) were added
30 36
3
7
Microwave Procedure. In an open microwave vial equipped with
a stirring bar, bis-alkyne 10 (26.5 mg, 0.048 mmol, 1 equiv), CuCl2·
to the stirring solution. EDC-HCl (900 mg, 4.70 mmol, 1.35 equiv)
was added last, and the mixture became bright yellow. The reaction
was warmed to 50 °C and stirred for 16 h. The crude mixture was
cooled back to room temperature, and PhMe and an aqueous solution
of citric acid (15 wt %) were added. The mixture was stirred for 5 min,
and the aqueous layer was discarded. Brine was added, and the
resulting mixture was stirred for an additional 5 min. The phases were
separated, and the organic phase was dried with MgSO4 and
concentrated in vacuo. The crude solid was azeotroped with PhMe,
and the clean alcohol was obtained as a colorless oil (1.23 g, 92%).
2
0
H O (2.0 mg, 0.012 mmol, 25 mol %), and Ni(NO ) ·6H O (3.5 mg,
2 3 2 2
.012 mmol, 25 mol %) were dissolved in MeOH (0.4 mL), and the
mixture was stirred at room temperature for 30 s or until the metals
were solubilized. Poly(ethylene) glycol 1450 (1.6 mL), TMEDA
(
0.035 mL, 0.24 mmol, 5 equiv), and Et N (0.02 mL, 0.144 mmol, 3
3
equiv) were added, and the mixture was stirred at room temperature
for an additional 30 s. The vial was then sealed with a microwave cap.
The reaction was warmed to 120 °C for 6 h. The crude mixture was
purified by chromatography (40 → 60% EtOAc in hexanes) to afford
the desired product as a white solid (13.7 mg, 50%).
2
5
1
[
5
α] = −67.3 (c = 0.0055, MeOH); H NMR (400 MHz, CDCl ) δ =
D
3
.49 (d, J = 9.1 Hz, 1 H), 4.69 (t, J = 8.7 Hz, 1 H), 4.60−4.51 (m, 1
Continuous Flow Procedure. In a 4 mL reaction vial equipped
with a stirring bar, bis-alkyne 10 (24.4 mg, 0.048 mmol, 1 equiv),
CuCl ·2H O (2.0 mg, 0.012 mmol, 25 mol %), and Ni(NO ) ·6H O
H), 4.27 (d, J = 8.9 Hz, 1 H), 4.06−3.97 (m, 2 H), 3.93−3.85 (m, 1
H), 3.79−3.73 (m, 1 H), 3.75 (s, 3 H), 2.41−2.31 (m, 1 H), 2.13 (s, 1
2
2
3
2
2
13
H), 2.09−1.98 (m, 1 H), 1.24 (s, 6 H), 1.06 (s, 9 H); C NMR (75
(
3.5 mg, 0.012 mmol, 25 mol %) were dissolved in MeOH (0.4 mL),
MHz, CDCl ) δ ppm = 172.4, 170.6, 156.4, 88.5, 71.8, 69.8, 69.0, 59.0,
3
and the mixture was stirred at room temperature for 30 s or until the
metals were solubilized. PEG cosolvent (1.6 mL), TMEDA (0.035 mL,
5
7.7, 56.3, 52.0, 37.3, 35.8, 31.5, 26.0, 25.5; HRMS (ESI) m/z
+
calculated for C H N O [M + H] , 383.2184; found, 383.2182.
19
31
2
6
0.24 mmol, 5 equiv), and Et N (0.02 mL, 0.144 mmol, 3 equiv) were
3
(3R,5S)-1-((S)-2-((((2,2-Dimethylbut-3-yn-1-yl)oxy)carbonyl)-
added, and the mixture was stirred at room temperature for an
additional 30 s and then taken up into a syringe. The reaction mixture
was injected using a 2 mL injection loop into the flow reactor for a
reaction time of 240 min (1 × 15 mL stainless steel reactor (tube-in-
amino)-3,3-dimethylbutanoyl)-5-(methoxycarbonyl)-
pyrrolidin-3-yl 4-ethynylisoindoline-2-carboxylate (10). Alco-
hol 9 (946 mg, 2.48 mmol, 1 equiv) and 1,1′-carbonyldiimidazole
(
(
CDI) (522 mg, 3.22 mmol, 1.3 equiv) were dissolved in dry DCM
12 mL). The mixture was stirred for 2 h at room temperature. Then,
tube, O (120 psi)) and 1 × 10 mL stainless steel reactor with a 32 cm
2
length section of stainless steel tubing between reactors) at a flow rate
of 0.104 mL/min at 120 °C. The flow reaction was conducted in a
Vapourtec R4 reactor and an R2+ pumping module. The continuous
flow setup is ended with a back pressure regulator (IDEX 250 psi).
Upon completion, silica gel was added to the collection flask and the
volatiles were removed under vacuum. The crude mixture was purified
by chromatography (40 → 60% EtOAc in hexanes) to afford the
desired product as a white solid (15.5 mg, 64%).
isoindoline (6) (579 mg, 3.22 mmol, 1.3 equiv) and Et N (1.03 mL,
3
7
.43 mmol, 3 equiv) were added, and the resulting mixture was
warmed to 50 °C and stirred for 18 h. Then the reaction was diluted
with DCM, and the phases were separated. The organic layer was
washed with HCl 1 M (2×), NaHCO3(satd), and brine. The organic
phase was dried with Na SO and concentrated in vacuo. Purification
2
4
by silica gel chromatography (40% EtOAc/hexanes gave the desired
2
5
bis-alkyne (1.25 g, 91%) as a white solid. Mp: 72.2 °C; [α] = −14.8
D
(c = 0.00135, MeOH). Note that rotamers are formed and can result in
1
ASSOCIATED CONTENT
complex splitting patterns in the H NMR, or can cause doubling of some
■
peaks in the 13C NMR spectrum. For clarity, all peaks are reported. H
1
*
S
Supporting Information
NMR (500 MHz, CDCl ) δ = 7.41−7.35 (m, 1H), 7.26−7.17 (m,
3
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.7b01308.
2
1
H), 5.43−5.37 (m, 2H), 4.83−4.61 (m, 4H); 4.24 (d, J = 9.5 Hz,
H), 4.20 (t, J = 12.5 Hz, 1H), 3.94−3.84 (m, 1H), 3.80 (d, J = 10.3
Hz, 1H), 3.77−3.75 (m, 3H), 3.55−3.46 (m, 1H), 3.32−3.27 (m, 1H),
Surface tension measurements, macrocyclization data in
tabular form, and spectroscopic data for all new
compounds (PDF)
2
1
.55−2.48 (m, 1H), 2.26−2.18 (m,1H), 2.06 (d, J = 7.5 Hz, 1H),
13
.26−1.22 (m, 1H), 1.13−1.06 (m, 6H), 1.05 (s, 9H); C NMR (125
MHz, CDCl ) δ ppm = 172.0, 171.05, 171.00, 156.3, 153.8, 153.7,
3
1
1
6
3
39.8, 139.6, 136.9, 136.5, 131.0, 130.9, 127.71, 127.67, 123.1, 122.9,
17.3, 117.2, 88.8, 88.7, 81.9, 81.5, 80.4, 73.5, 71.68, 71.65, 68.73,
8.68, 59.2, 59.1, 57.9, 57.8, 54.03, 53.95, 53.0, 52.5, 52.3, 52.0, 35.4,
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: shawn.collins@umontreal.ca.
5.3, 35.0, 34.8, 31.5, 31.4, 26.2, 25.6, 25.5; HRMS (ESI) m/z
+
calculated for C H N O [M + H] , 552.2710; found, 552.2704.
30
38
3
7
F
DOI: 10.1021/acs.joc.7b01308
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
ORCID
287. (e) Ouyang, C.; Chen, S.; Che, B.; Xue, G. Colloids Surf., A 2007,
3
(
(
1
01, 346−351.
10) Bedard, A.-C.; Collins, S. K. ACS Catal. 2013, 3, 773−782.
11) Yin, W.; He, C.; Chen, M.; Zhang, H.; Lei, A. Org. Lett. 2009,
1, 709−712.
12) Clinically approved PEGylated products: Adagen, Oncaspar,
Shawn K. Collins: 0000-0001-9206-5538
Notes
The authors declare no competing financial interest.
́
(
ACKNOWLEDGMENTS
The authors acknowledge the Natural Sciences and Engineering
Research Council of Canada (NSERC), the NSERC CREATE
Doxil, PEGasys, PegIntron, Neulasta, Somavert, Macugen, Mircera,
Cimzia, Sylatron. See: Mishra, P.; Nayak, B.; Dey, R. K. Asian J. Pharm.
Sci. 2016, 11, 337−348.
■
(13) Thakur, S.; Kesharwani, P.; Tekade, R. K.; Jain, N. K. Polymer
́ ́
program in Continuous Flow Science, Universite de Montreal,
2
(
015, 59, 67−92.
and the Centre for Green Chemistry and Catalysis (CGCC) for
generous funding. The Canadian Foundation for Innovation is
thanked for the funding of continuous flow infrastructure.
Vanessa Kairouz is thanked for assistance and valuable advice
14) See the Supporting Information for details.
(15) Note that all data are presented in tabular format in the
Supporting Information.
(16) For an alternative route to alkynyl alcohol 4, see: Trost, B. M.;
Hu, Y.; Horne, D. B. J. Am. Chem. Soc. 2007, 129, 11781−11790.
(17) Previous reports have not shown any decrease in efficiency of
the phase separation system in continuous flow. See ref 18 for details.
́
concerning continuous flow protocols. E.G., A.-C.B., and M.R.
thank NSERC and the FRQNT for graduate scholarships.
(
1
(
18) Bed
962−1966.
19) (a) Petersen, T. P.; Polyzos, A.; O’Brien, M.; Ulven, T.;
Baxendale, I. R.; Ley, S. V. ChemSusChem 2012, 5, 274−277.
b) Brzozowski, M.; O’Brien, M.; Ley, S. V.; Polyzos, A. Acc. Chem.
Res. 2015, 48, 349−362.
20) For example, proline units are often found in macrocyclic
́ ́
ard, A.-C.; Regnier, S.; Collins, S. K. Green Chem. 2013, 15,
REFERENCES
■
(
1) (a) Roxburgh, C. J. Tetrahedron 1995, 51, 9767−9822.
(
b) Marsault, E.; Peterson, M. L. J. Med. Chem. 2011, 54, 1961−
2
2
2
004. (c) Matsuda, H.; Watanabe, S.; Yamamoto, K. Chem. Biodiversity
004, 1, 1985−1991. (d) Ruedi, G.; Nagel, M.; Hansen, H.-J. Org. Lett.
004, 6, 2989−2991. (e) Fehr, C.; Galindo, J.; Etter, O.; Thommen,
(
̈
(
W. Angew. Chem., Int. Ed. 2002, 41, 4523−4526.
2) For a chart illustrating the interest in macrocycles in both drug
discovery and total synthesis through the relative number of citations,
see the Supporting Information.
3) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. Nat. Rev. Drug
Discovery 2008, 7, 608−624.
4) (a) Seiple, I. B.; Zhang, Z.; Jakubec, P.; Langlois-Mercier, A.;
Wright, P. M.; Hog, D. T.; Yabu, K.; Allu, S. R.; Fukuzaki, T.; Carlsen,
P. N.; Kitamura, Y.; Zhou, X.; Condakes, M. L.; Szczypinski, F. T.;
scaffolds as they are known to promote β-turn mimics and influence
macrocyclization reactions. See: Chouhan, G.; James, K. Org. Lett.
(
2
013, 15, 1206−1209. For an example of the structure of a linear
precursor for macrocyclization being predisposed to macrocyclization
by a certain mode of catalysis, while being unfavorable toward
cyclization using an alternate mode of catalysis, see: Beckmann, H. S.
G.; Nie, F.; Hagerman, C. E.; Johansson, H.; Tan, Y. S.; Wilcke, D.;
Spring, D. R. Nat. Chem. 2013, 5, 861−867.
(
(
́
(
21) It was not possible to identify a dimer formed from Glaser−Hay
coupling of the more reactive aryl alkyne.
22) When the bis-alkyne 10 was subjected to macrocyclization in 2:1
Green, W. D.; Myers, A. G. Nature 2016, 533, 338−345. (b) Nie, F.;
Kunciw, D. L.; Wilcke, D.; Stokes, J. E.; Galloway, W. R. J. D.; Bartlett,
S.; Sore, H. F.; Spring, D. R. Angew. Chem., Int. Ed. 2016, 55, 11139−
(
StarPEG1014:MeOH, only a 31% yield of macrocycle 11 was obtained
with complete conversion of the starting bis-alkyne 10. See the
Supporting Information.
1
1143. (c) Collins, S.; Bartlett, S.; Nie, F.; Sore, H. F.; Spring, D. R.
Synthesis 2016, 48, 1457−1473.
5) (a) Harper, S.; McCauley, J. A.; Rudd, M. T.; Ferrara, M.;
(
(
∼
(
2
(
2
(
23) In continuous flow, a throughput of ∼100 mg/d (24 mM) or
230 mg/d (100 mM) is estimated.
24) Macrocyclization at 200 mM (4:1 PEG400:MeOH) afforded only
1% of the desired macrocycle 11.
25) Haley, M. M.; Pak, J. J.; Brand, S. C. Top. Curr. Chem. 1999,
01, 81−130.
26) For an elegant example of phase separation used to minimize
DiFilippo, M.; Crescenzi, B.; Koch, U.; Petrocchi, A.; Holloway, M. K.;
Butcher, J. W.; Romano, J. J.; Bush, K. J.; Gilbert, K. F.; McIntyre, C.
J.; Nguyen, K. T.; Nizi, E.; Carroll, S. S.; Ludmerer, S. W.; Burlein, C.;
DiMuzio, J. M.; Graham, D. J.; McHale, C. M.; Stahlhut, M. W.; Olsen,
D. B.; Monteagudo, E.; Cianetti, S.; Giuliano, C.; Pucci, V.; Trainor,
N.; Fandozzi, C. M.; Rowley, M.; Coleman, P. J.; Vacca, J. P.; Summa,
V.; Liverton, N. J. ACS Med. Chem. Lett. 2012, 3, 332−336. (b) Kong,
J.; Chen, C.-y.; Balsells-Padros, J.; Cao, Y.; Dunn, R. F.; Dolman, S. J.;
Janey, J.; Li, H.; Zacuto, M. J. J. Org. Chem. 2012, 77, 3820−3828.
the concentration of hazardous intermediates, see: (a) Morandi, B.;
Carreira, E. M. Science 2012, 335, 1471−1474. (b) Morandi, B.;
Carreira, E. M. Angew. Chem., Int. Ed. 2010, 49, 938−941.
̈
c) Ali, A.; Aydin, C.; Gildemeister, R.; Romano, K. P.; Cao, H.; Ozen,
A.; Soumana, D.; Newton, A.; Petropoulos, C. J.; Huang, W.; Schiffer,
(
(
27) Shriver, D. F.; Drezdzon, M. A. The Manipulation of Air-Sensitive
Compounds; Wiley-VCH: New York, 1986.
28) Chen, G.-Q.; Zhang, X.-N.; Wei, Y.; Tang, X.-Y.; Shi, M. Angew.
Chem., Int. Ed. 2014, 53, 8492−8497.
29) Maison, W.; Arce, E.; Renold, P.; Kennedy, R. J.; Kemp, D. S. J.
Am. Chem. Soc. 2001, 123, 10245−10254.
30) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923−
925.
C. A. ACS Chem. Biol. 2013, 8, 1469−1478.
(
(
6) (a) Bed
9976. (b) Bed
420−6422. (c) Bed
108−2113.
́
ard, A.-C.; Collins, S. K. J. Am. Chem. Soc. 2011, 133,
1
6
2
́
ard, A.-C.; Collins, S. K. Chem. Commun. 2012, 48,
ard, A.-C.; Collins, S. K. Chem. - Eur. J. 2013, 19,
(
́
(
2
(
́
7) (a) Anastas, P. T. ChemSusChem 2009, 2, 391−392. (b) Horvath,
I. T.; Anastas, P. T. Chem. Rev. 2007, 107, 2169−2173. (c) Constable,
D. J. C.; Curzons, A. D.; Cunningham, V. L. Green Chem. 2002, 4,
5
21−527. (d) Curzons, A. D.; Constable, D. J. C.; Mortimer, D. N.;
Cunningham, V. L. Green Chem. 2001, 3, 1−6.
8) Ito, H.; Mitamura, Y.; Segawa, Y.; Itami, K. Angew. Chem., Int. Ed.
015, 54, 159−163.
9) Surface tension measurements are well-precedented for the
(
2
(
analysis of PEG aggregates; for examples, see: (a) Dey, J.; Shrivastava,
S. Soft Matter 2012, 8, 1305−1308. (b) Yang, S.-C.; Faller, R. Langmuir
2
012, 28, 2275−2280. (c) Alam, M. S.; Mandal, A. B. J. Mol. Liq. 2012,
1
68, 75−79. PEG aggregates can also be analyzed by the use of UV
and IR spectroscopy; see: (d) Froehlich, E.; Mandeville, J. S.; Arnold,
D.; Kreplak, L.; Tajmir-Riahi, H. A. Biomacromolecules 2012, 13, 282−
G
DOI: 10.1021/acs.joc.7b01308
J. Org. Chem. XXXX, XXX, XXX−XXX