Development of a Large-Scale Route to Glecaprevir: Synthesis of the Macrocycle via Intramolecular Etherification

Article abstract of DOI:10.1021/acs.oprd.0c00244

Glecaprevir was identified as a potent hepatitis C virus (HCV) protease inhibitor, and a large-scale synthesis was required to support the late-stage clinical trials and subsequent commercial launch. The large-scale synthetic route to glecaprevir required the development of completely new synthetic approaches to the two key structural features: the 18-membered macrocycle 3 and the difluoromethyl-substituted cyclopropyl amino acid 4. In this first manuscript, we describe the route development for the macrocycle 3; the second manuscript will describe the development of a new synthetic route to the difluoromethyl-substituted cyclopropyl amino acid 4 and the final assembly of glecaprevir. The large-scale synthetic route to the macrocycle employed a unique intramolecular etherification reaction as the key step in the macrocycle synthesis, avoiding the scalability limitations of the ring-closing metathesis (RCM) reaction of the enabling route. The large-scale synthetic route to the macrocycle was successfully used to produce the amount of glecaprevir required to support the late-stage clinical development.

Full text of DOI:10.1021/acs.oprd.0c00244

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Full Paper
Development of a Large-Scale Route to Glecaprevir:
Synthesis of the Macrocycle via Intramolecular Etherification
Jeffrey M. Kallemeyn, Kenneth Michael Engstrom, Matthew Pelc, Kirill Lukin, Westin Morrill, Haojuan
Wei, Timothy Towne, Jeremy Henle, Nandkishor Nere, Dennie S Welch, Shashank Shekhar, Matthew
Ravn, Gang Zhao, Michael G. Fickes, Chen Ding, John (Cody) Vinci, James Marren, and Russell D Cink
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00244 • Publication Date (Web): 13 Jul 2020
Downloaded from pubs.acs.org on July 13, 2020
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Development of a Large-Scale Route to Glecaprevir:
Synthesis of the Macrocycle via Intramolecular
Etherification
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Jeffrey M. Kallemeyn, Kenneth M. Engstrom, Matthew J. Pelc, Kirill A. Lukin, Westin H.
Morrill, Haojuan Wei, Timothy B. Towne, Jeremy Henle, Nandkishor K. Nere, Dennie S. Welch,
Shashank Shekhar, Matthew M. Ravn, Gang Zhao, Michael G. Fickes, Chen Ding, John C. Vinci,
James Marren, and Russell D. Cink*
Process Research & Development, and Analytical Research & Development, AbbVie Inc., 1401
Sheridan Road, North Chicago, IL, 60064
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For Table of Contents Only (first image is TIFF at 300 dpi, second is chemdraw)
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Key Step to Macrocycle
Intramolecular Etherification
Glecaprevir
KEYWORDS
glecaprevir, ABT-493, HCV, intramolecular etherification, macrocycle
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Abstract: Glecaprevir was identified as a potent HCV protease inhibitor and a large-scale
synthesis was required to support the late stage clinical trials and subsequent commercial launch.
The large-scale synthetic route to glecaprevir required the development of completely new
synthetic approaches to the two key structural features: the 18-membered macrocycle 3, and the
difluoromethyl substituted cyclopropyl amino acid 4. In this first manuscript, we describe the
route development for the macrocycle 3; the second manuscript will describe the development of
a new synthetic route to the difluoromethyl substituted cyclopropyl amino acid 4 and final
assembly of glecaprevir. The large-scale synthetic route to the macrocycle employed a unique
intramolecular etherification reaction as the key step in the macrocycle synthesis, avoiding the
scalability limitations of the RCM reaction of the enabling route. The large-scale synthetic route
to the macrocycle was successfully used to produce the amount of glecaprevir required to
support the late stage clinical development.
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Introduction
Glecaprevir was identified as a potent HCV NS3/4A protease inhibitor, active against all major
HCV genotypes.1 Glecaprevir was developed in combination with pibrentasvir, an NS5A
inhibitor which also shows pan-genotypic activity. An enabling synthesis to glecaprevir was
employed to support the pre-clinical development and subsequent Phase I clinical trials.2 The
enabling synthesis was suitable to produce the material required for early development, but
suffered from two limitations that would hamper the large-scale production required for late
stage clinical development.
The initial disconnection in the retrosynthetic analysis for both the enabling route and the
large-scale route for glecaprevir (1), shown in Scheme 1, dissects glecaprevir into amino
sulfonamide 2 and macrocycle 3. The first limitation of the enabling synthesis was the efficiency
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of the ring closing metathesis (RCM) process used to form the macrocycle. The second
limitation of the enabling route was the efficiency of the synthesis of the difluoromethyl
substituted cyclopropyl amino acid 4. Based on the scale limitations of the enabling route, it was
clear that new syntheses for macrocycle 3 and amino acid 4 would be required for large scale
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production. The focus of this manuscript is the development of a new route to macrocycle 3;
the new route to amino acid 4 and the final assembly of glecaprevir are the subject of the
following manuscript.4
Scheme 1: Retrosynthetic Analysis to Glecaprevir
F
F
F
F
N
N
N
N
O
O
O
O
O
O
O O
S
H
O
O O
S
H
H2N
N
O
N
Boc
OH
N
N
O
N
N
OH
H
O
H
O
O
O
F
F
O
O
N
H
F
F
F
N
H
F
2
4
Glecaprevir, 1
3
The synthetically challenging structural features to be addressed in the development of a new
synthesis for macrocycle 3 are highlighted in red in Scheme 1. A new synthesis of macrocycle 3
posed two related synthetic challenges: a new approach to close the macrocycle, and the
construction of the red highlighted six-carbon section of the macrocycle. This six-carbon section
contains considerable structural complexity, including an ether, a trans alkene, a gem-
difluoromethylene, and a quinoxaline. The primary synthetic challenge was deemed to be
installation of the gem-difluoromethylene. Significant advances have recently occurred in the
5
synthesis of substrates containing a gem-difluoromethylene. With these challenges in mind, the
development of the large-scale synthetic route to macrocycle 3 began.
Results and Discussion
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Potential Alternate Routes to the Macrocycle
While multiple approaches to form the macrocycle were considered, the two main
disconnection points evaluated, options A and B, are shown in Scheme 2. The appeal of option A
was that multiple approaches were possible to form the macrocycle through the
bromodifluoroquinoxalines 5-8. The alkene 5 could undergo an intramolecular Heck reaction to
form the macrocycle.6 The alkyne 6 could undergo a Sonogashira type reaction followed by
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selective reduction to form the trans alkene. Both the alkene 5 and the alkyne 6 could undergo
8
a photoredox catalyzed radical cyclization to form the macrocycle. The alkyne 6 could also be
converted to the boronic acid 7 or the boronate ester 8 followed by an intramolecular Suzuki
reaction to form the macrocycle.9 One could also envision switching the order of the bond
forming steps, first forming the carbon-carbon bond in an intermolecular coupling and then
closing the macrocycle through the lactam. Option B in Scheme 2 involved an intramolecular
etherification of the allylic bromide 12.10 While etherification with an allylic bromide would
normally be considered a straightforward reaction, the probability of success in the formation an
1
8-membered macrocycle was uncertain.
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Scheme 2: Potential Options to Form the Macrocycle
R
F
F
F
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N
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F F
Br
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Br
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Option A
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O
H
N
H
3
5
= alkene
6 = alkyne
11
9 = alkene
10 = alkyne
7
= vinyl boronic acid
8
= vinyl boronate ester
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Br
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Br
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O
O
N
OH
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OH
O
Option B
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O
+
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OH
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OMe
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O
O
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N
H
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H
3
14
13
1
2
The second key consideration for the synthesis of the macrocycle was the source of the gem-
difluoromethylene within the six-carbon section highlighted in Scheme 1. Routes that involved
fluorination of an advanced intermediate were deemed impractical due to the cost, handling, and
limited availability of many fluorination reagents on scale. Instead, the strategy was to develop
routes starting from fluorinated building blocks that were commercially available on large-scale.
The readily available fluorinated starting material ethyl trifluoropyruvate 17 contained the most
complete section of the carbon backbone, as shown Scheme 3 for the retrosynthetic analysis
from macrocycle 3. Elaboration of 17 would involve formation of the requisite carbon-carbon
bond with defluorination.
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Scheme 3: Retrosynthesis to Fluorinated Starting Material
F
F
N
N
F
F
O
O
O
H2N
H2N
O
R
RO
O
N
OH
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EtO
O
17
Evaluation of Option A to the Macrocycle
Most of the reactions under consideration for option A in Scheme 2 employed palladium
catalysis in the presence of ligand and base. There were significant concerns regarding the
stability of the allylic difluoride functionality under these conditions. A stability test of the
macrocycle ester 18 to palladium catalysis conditions, shown in Table 1, confirmed these
concerns with significant decomposition due to loss of HF in the presence of palladium and
ligand (entries 4 and 5). The key challenge for the palladium catalyzed reactions would be
identifying conditions in which the desired carbon-carbon forming reaction proceeded
significantly faster than the side reaction leading to product decomposition.
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Table 1. Stability of Macrocycle Ester 18 to Pd Catalysis Conditions
F
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F
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O
O
O
Conditions
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O
N
OMe
DME/water
60 °C
N
OMe
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18
Pd
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(dba)
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RuPhos
K
3
PO
4
Time
Ester 18
Entry
(equiv)
(equiv) (equiv)
(h) HPLC Area %
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0.05
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100
92 a
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(
a) Low levels of ester hydrolysis and epimerization observed.
In order to evaluate the macrocycle synthesis through the option A pathways, the synthesis of
bromodifluoroquinoxaline 11 was developed, as outlined in Scheme 4. Condensation of ethyl
trifluoropyruvate 17 with diaminobenzene 16 produced quinoxaline 19 in near quantitative yield.
The hydrogenation of 19 followed by base treatment proved to be an efficient method to convert
the trifluoromethyl quinoxaline 19 to the difluoromethyl quinoxaline 21. Attempts at alkylation
by deprotonation of the difluoromethyl of quinoxaline 22 and related substrates proved to be
problematic. The electron withdrawing difluoromethyl group caused the  carbon within the
quinoxaline to be exceptionally electrophilic, resulting in dimerization of 22 during
deprotonation. Interestingly, the only electrophile identified that could be cleanly trapped by the
anion of quinoxaline 22 was brominating agent 23; facilitating the unique bromination for the
conversion of difluoromethyl quinoxaline 22 to the bromodifluoromethyl quinoxaline 24. Slow
addition of LiHMDS (3 equiv) to a solution of quinoxaline 22 and bromine (2 equiv) induced in
situ formation of brominating agent 23, followed by deprotonation of the difluoromethyl group
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and trapping of the resulting anion by 23. The synthesis of 11 was completed by S Ar reaction of
N
quinoxaline 24 with hydroxyproline 25 followed by deprotection. The overall yield of 66% for
the seven-step sequence to produce 11 was very encouraging for this approach.
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Scheme 4: Synthesis of Bromodifluoroquinoxaline 11
H N
2
F
F
O
H
N
F3C
HO
N
F3C
HO
N
H N
OEt
2
H , Pd/C
2
NaOH
H
SOCl2
F3C
16
O
THF
N
N
THF, 50 °C
HO
N
i-PrOAc
THF, 45 °C
50 °C
DMF, 60 °C
97%
17
19
20
21
three steps
89%
HO
F
F
N
N
Br
CO Me
2
F
F
N
Boc
F
F
N
N
O
LiHMDS, Br2
H
N
25
Br
THF, 0 °C
Cl
1
) Cs CO , DMF
2 3
Cl
N
HN
Br
N
2
) HCl, MeOH
HCl CO Me
22
2
24
Me Si
SiMe3
76%
11
3
from 22
23
Completion of the synthesis of the substrates for evaluation of the option A cyclization
strategies followed the general strategy outlined in Scheme 2.11 A significant amount of
screening was conducted for the cyclization methods of option A, and selected results are
summarized in Scheme 5. Screening of the intramolecular Heck reaction of alkene 5 (Scheme 2)
revealed the main product formed was consistent with cyclization followed by loss of HF. A
similar result was observed in the intramolecular Sonogashira type reaction of alkyne 6;
however, conversion was low. The intramolecular Suzuki reaction of boronate ester 8, shown in
Scheme 5, was screened extensively. The best results were obtained with sterically hindered
phosphine ligands such as P(t-Bu) and RuPhos, but the yield reached a maximum of 11% due to
3
significant product decomposition from loss of HF.12 The boronic acid 7 did not offer any
improvement. The intramolecular photoredox catalyzed cyclization reaction of alkene 5 was not
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successful, but a low yield of 18 was observed using the boronic acid 7. The best result for the
photoredox cyclization was obtained with alkyne 6, shown in Scheme 5, which produced a 30%
yield of macrocycle 18 in a 2:1 ratio of trans/cis alkene isomers. A model vinyl boronate ester 26
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3
4
5
6
7
8
9
0
was used to evaluate the intermolecular Suzuki reaction with bromodifluoroquinoxaline 27.13
Under nickel catalysis, negligible cross coupling occurred, along with up to 10% of
homocoupling of 27.14 Under palladium catalysis, the reaction showed rapid conversion to the
coupled product 28, but also significant decomposition through loss of HF. The addition of CsF
(2 equiv) was found to minimize impurities from the loss of HF to approximately 5%, and the
addition of water (1 equiv) resulted in faster conversion and improved product yield. The best
result obtained for the intermolecular Suzuki coupling was a 62% assay yield for 28,
demonstrating the feasibility of this approach to form the desired carbon-carbon bond. The
challenge for this approach would be the narrow operating window between achieving reaction
completion and over reaction with loss of HF resulting in erosion of the product yield. This
intermolecular Suzuki approach, in combination with subsequent macrocycle closure through the
lactam, was deemed a feasible approach to form the macrocycle; but the product stability under
the cross-coupling reaction conditions was recognized as a liability for large-scale production.
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Scheme 5: Select Results for Option A Trials
O
F
F
B
O
N
N
F
F
N
N
O
Br
O
O
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
O
N
OMe
O
N
OMe
11% yield
O
O
O
N
H
O
O
N
H
18
8
F
F
F
F
N
N
N
Br
O
O
N
O
O
b
O
N
OMe
O
N
OMe
30% yield
O
O
2:1 trans:cis
O
O
O
O
N
N
H
H
1
8
6
F
F
F
F
O
B
N
N
N
O
Br
O
O
N
O
c
OPr
BocN
O
BocN
OMe
62% yield
OMe
OPr
O
O
28
26
27
Reagents and Conditions: (a) Pd (dba) (1.5 mol%), RuPhos (3.6 mol%),
2
3
K PO (2 eq), DME/water (20:1), 50 °C. (b) Ru(bpy) (1 mol%),
3
4
3
CH (CH ) SH (1.5 eq), DIPEA (2 eq), 4:1 toluene:CH CN, 32 °C, blue
3
2 11
3
LEDs. (c) Pd (dba) (1.5 mol%), RuPhos (3.6 mol%), K PO (2 eq),
2
3
3
4
CsF (2 eq), H O (1 eq) DME, 50 °C.
2
Evaluation of Option B to the Macrocycle
In order to test the option B cyclization shown in Scheme 2, the synthesis of allylic bromide 12
was undertaken starting with intermediates available from the RCM reaction of the enabling
route.15 As shown in entry 1 of Table 2, the initial cyclization experiment utilized 40% aqueous
benzyltrimethylammonium hydroxide (Triton® B) as base and revealed the cyclization
proceeded cleanly; yielding the desired macrocycle 3 and a lower level of impurity 29.
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Interestingly, S 2ʹ cyclization was not observed; rather impurities 29 and 30 were formed
N
through cyclization of the carbamate.16 A solvent screen (entries 2-5) revealed that the use of a
biphasic toluene/water system produced a 74% combined assay yield for the macrocycle 3 and
the macrocycle ester 18, with very low levels impurities 29 and 30. This result demonstrated the
feasibility of the intramolecular etherification approach to form the macrocycle. In fact, the assay
yield for this early result was already superior to the RCM reaction of the enabling route.2
Additionally, the product was isolated in > 99.5% purity upon crystallization. With these results
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
in hand, the focus shifted to developing the synthesis of allylic bromide 12.
Table 2. Initial Cyclization Results of Option B
F
F
F
F
Br
N
N
N
N
N
F
N
O
OH
O
F
O
O
O
Conditions
O
O
HO
N
OMe
N
OR
O
O
O
N
N
OR
O
O
O
O
O
N
H
N
H
O
12
3 R= H
8 R= Me
29 R= H
0 R= Me
1
3
Solvent
Volumes
BnMe
3
NOH Time
Assay
Yield %a
HPLC Area %
Entry
Solvent
(equiv.)
2.8
(h)
13
2.7
20
16
15
3
18
29
30
1
2
3
4
5
THF
NMP
10
10
10
20
30
51
-
57
-
-
-
29
6
-
76
-
1.2
MTBE
Toluene
1.2
39
57
74
72
58
78
-
10
8
2.6
10
4
4
Toluene/water (2:1)
10
5
1
(
a) Combined assay of 3 and 18.
Retrosynthesis of Allylic Bromide 12
The retrosynthesis of allylic bromide 12, shown in Scheme 6, initially dissects the amide bond
of 12 into carbamate 13 and allylic bromide 14. The carbamate 13 could be synthesized from
alkene 9, an intermediate in the enabling route to glecaprevir.2 To minimize potential side
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reactions from carrying allylic bromide 14 through multiple steps, the alcohol 31 was considered
as a potential masking group for subsequent conversion to the bromide. Intermediates 14 or 31
could be derived through an S Ar reaction of a protected hydroxyproline with quinoxaline 32
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
followed by an allylic bromination or oxidation with transposition of the alkene. Quinoxaline 32
could be readily prepared from the known keto ester 33.17 The majority of steps in the
retrosynthesis to 12 were perceived to have a high probability of success, but the allylic
bromination or oxidation with alkene transposition was considered to be challenging due to the
presence of the gem-difluoromethylene.
Scheme 6: Retrosynthesis of Allylic Bromide 12
F
F
F
F
R
N
N
Br
N
N
OH
OH
O
O
O
O
OH
O
HN
OMe
N
OMe
O
O
N
H
O
O
O
N
H
14 R = Br
31 R = OH
13
12
F
F
F
F
O
O
N
EtO
Cl
N
33
32
Synthesis of Carbamate 13
The synthesis of carbamate 13, shown in Scheme 7, was based on the synthesis of alkene 9
2
which was previously described in the enabling route to glecaprevir. The isolation of 9 as the
dicyclohexylamine salt was eliminated from the process, and the subsequent deallylation of 9
proceeded as expected, with carbamate 13 isolated as the diisopropylamine (DIPA) salt. The
robust six-step sequence produced carbamate 13 in 26% yield from cyclopentene oxide 34.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 7: Synthesis of Carbamate 13
(
R)
Ac O,
DMAP
2
O
HO
Novozym 435
aqueous
O
O
35
38
(R)
(S)
OH
O
DIPEA,
Heptane, 0 °C
BF OEt
OAc
(±)
3
2
OH
(±)
KH PO4
2
Toluene
to 20 °C
9%
K2HPO4
5
(S)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
34
O
6
36
37
47% from 36
39
>
99.8% ee
OAc
(R)
O
1) triphosgene, 2,6-lutidine
toluene, - 5 °C
O
1) Pd/C, MeOH
aq. HCl, 50 °C
OH
O
38
(R)
OH
O
O
O
CO H
CO2HHN
2
2) NaOH,
water
CO2H
2) DIPA, i-PrOAc
heptane
HN
HN
H2N
(S)
40
O
5 to 20 °C
39
79% from 38
(S)
OAc
9
13
Synthesis of Quinoxaline 32
The synthesis of quinoxaline 32, shown in Scheme 8, began with the known synthesis of keto
ester 33.17 While the route to 33 was already established, some unique aspects of the large-scale
synthesis are worth discussion. The starting pyruvate 17 contains a low level of the
corresponding fluorohydrin impurity which can release HF upon reaction with allyl alcohol. To
avoid the potential for HF corrosion, pyridine was charged to the reactor prior to the addition of
1
7, and upon completion the reaction was quenched into aqueous base to avoid an acidic pH
environment. To ensure a reproducible activation of Zn with TMSCl, the oxygen level was
controlled below 50 ppm and the slurry mixed for at least 90 minutes after the TMSCl addition.
A portion-wise addition of the activated Zn slurry to chloride 41 was important to control the
reaction temperature and minimize impurities formed by over-reduction. The reactor set up,
shown in Figure 1, utilized recirculation of the Zn slurry in DMF with the return diptube near the
bottom of the reactor and angled to create a vortex. This facilitated nearly complete transfer of
the slurry when the volume dropped below the agitator. The slurry was recirculated throughout
the deoxygenation and activation, and subsequently portions of the Zn slurry were added to the
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second reactor containing chloride 41. After filtration of the slurry, the solution was heated to
induce the Claisen rearrangement and followed by condensation with diaminobenzene 16 to form
the quinoxaline 43. The Zn reduction also generates fluoride and consequently was also a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
potential source of corrosion. Quenching the condensation reaction with aqueous NH Cl induced
4
crystallization of quinoxaline 43, ensured solubility of the Zn salts, and produced a mixture that
was not corrosive to stainless steel. A straightforward chlorination and subsequent crystallization
completed the synthesis of quinoxaline 32.
Scheme 8: Synthesis of Quinoxaline 32
SOCl2
pyridine
O
O
Cl
O
Zn dust
OEt TMSCl
OEt CH2Cl2
OH
F3C
F3C
O
0°C
DMF
35
0
to 15 °C
17
41
85%
NH2
O
O
80 °C
OEt
NH2
F
OEt
16
F
F
O
F
O
33
65%, 3 steps
42
F
F
F
F
N
POCl3
N
Toluene
DMF
HO
N
Cl
N
50 °C
43
32
90%
Figure 1. Reactor Configuration for Zn Reduction
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Methods for Allylic Functionalization with Transposition
Numerous methods were evaluated to perform the allylic functionalization with transposition
on quinoxaline 32 and similar substrates. Mild halogenation conditions resulted in no conversion,
whereas forcing conditions to activate the alkene induced intramolecular attack of the
quinoxaline nitrogen to form 5-membered cyclic impurities. The allylic oxidation conditions of
White were successful, but the product yield and purity were too low for large-scale
production.18 Ultimately, epoxidation and dihydroxylation were the only methods found to
cleanly oxidize the alkene of quinoxaline 32 and related substrates. As outlined in Scheme 9, the
epoxide 44 or the diol 45 could be converted to the allylic alcohol 31. As outlined in Scheme 6,
allylic bromide 14 or allylic alcohol 31 were possible intermediates in the synthesis of 12. Based
on the required intermediacy of allylic alcohol 31, the strategy was to conduct the subsequent
conversion to the allylic bromide after the coupling of 31 with carbamate 13. Both the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
epoxidation and dihydroxylation approaches were evaluated for the synthesis of the macrocycle.
Scheme 9: Options for Allylic Oxidation with Transposition
F
F
O
F
F
N
N
HO
N
N
F
F
R
N
N
O
44
Cl
OH F
F
HN
OMe
32
HO
N
N
O
3
1
R
45
R = Cl or hydroxyproline
Epoxidation Route to Allylic Alcohol 31
The epoxidation route began with the S Ar reaction of quinoxaline 32 with hydroxyproline 46
N
using NaH as the base.19 Numerous bases were screened for the synthesis of ether 47;
elimination of HF was a significant side reaction resulting in low yield and purity for bases other
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than NaH. After considerable development, the epoxidation method of Yang was successfully
applied to synthesize epoxide 48.20 The epoxide was unstable under acidic conditions, requiring
a careful workup and isolation as a solution of the sodium salt. The epoxide ring opening
performed acceptably with LiHMDS, however potassium tert-amylate (KOtAm) provided a
slightly improved yield. Elimination of HF was also a side reaction in the epoxide opening,
resulting in oligomer formation that required a DMEDA quench and complex workup to yield a
stable product solution. The epoxide opening also produced 7.2% of the cis alkene isomer
impurity. After considerable screening, crystallization of 49 as the benzhydrylamine
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Ph CHNH ) salt was identified and facilitated isolation of allylic alcohol 49 in 63% yield. After
2 2
a salt break, treatment of allylic alcohol 49 with a solution of thionyl chloride in methanol
allowed simultaneously esterification of the carboxylic acid and deprotection of the amine
resulting in clean formation of 31 in high yield and purity.
Each of the first three steps in Scheme 10 had liabilities for large-scale production: the use of
NaH in the first step; the throughput, Oxone® waste, and product stability in the second step;
and the modest yield and impurity formation in the third step. The epoxidation route produced
allylic alcohol 31 in 40% overall yield from quinoxaline 32, but the above-mentioned liabilities
were recognized as a challenge for continued large-scale production.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 10: Epoxidation Route to Allylic Alcohol 31
F
F
F F
O
N
N
N
N
1) EtOAc, aq. HCl
2) Oxone®
OH
F
F
O
O
1
) NaH, THF
N
N
5
to 20 °C
CF COCH
3
3
BocN
BocN
BocN
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
Cl
2) DIPA, MTBE
heptane
HN
aq. NaHCO3
CO H
2
CH CN, 0 °C
OH
3
ONa
O
O
46
32
90%
75%
47
48
F
F
HO
N
N
F
F
HO
N
N
O
1) KOt-Am, Toluene, -10 °C
2
) DMEDA, MeOH, aq. H PO4
O
1) CPME, aq. H3PO4
3
48
HN
HCl
3
) Ph CHNH , i-PrOAc
2) SOCl2, MeOH
CPME, -10 to 20 °C
2
2
BocN
OMe
Ph
Ph
O
63%
OH H2N
95%
O
31
49
Dihydroxylation Route to Allylic Alcohol 31
The dihydroxylation route to allylic alcohol 31 is shown in Scheme 11. The dihydroxylation of
quinoxaline 32 was found to be very robust and required only 0.15 mol% of potassium osmate
dihydrate catalyst and aqueous NMO as the oxidant.21 The subsequent conversion to the cyclic
carbonate 50 with CDI was also robust resulting in a 90% overall yield from 32. The conversion
of cyclic carbonate 50 to allylic alcohol 49 was accomplished in a two-step, one-pot process.
Treatment of 50 with NaHMDS at –5 °C afforded the open carbonate 51¸ which serves as a
transient protecting group for the allylic alcohol. We were pleasantly surprised to find that the
open carbonate 51 was sufficiently stable to allow the S Ar reaction to proceed cleanly upon
N
addition of a slurry of the dianion of hydroxyproline 46. Another positive observation for the
carbonate opening was the level of the cis alkene isomer was only 0.5%, a significant reduction
from the 7.2% observed in the epoxide opening. After an aqueous workup and carbon treatment,
allylic alcohol 49 was isolated by crystallization as the benzhydrylamine salt.
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Scheme 11: Dihydroxylation Route to Allylic Alcohol 31
O
O
F
F
F F
1) K OsO , NMO
EtOAc, 40 °C
O
F
F
O
O
N
N
N
N
2
4
N
N
1) NaHMDS
O
Cl
Cl
2) CH CN, CDI, 40 °C
3
THF
-5 °C
Cl
50
32
51
90%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
F F
F
F
HO
N
HO
N
BocN
O
N
O
N
CO H
2
46
1) CPME, aq. H PO
3
4
51
HN
HCl
BocN
2) KOtBu, THF, -5 °C
2) SOCl2, MeOH
Ph
Ph
3
) Ph CHNH , i-PrOAc
CPME, -10 to 20 °C
OMe
2
2
OH H2N
O
O
69%
95%
3
1
49
A variety of bases were screened for the carbonate opening and NaHMDS and LiHMDS gave
the cleanest profiles. The alkoxide bases (NaOtBu, KOtBu, KOtAm) all gave numerous
impurities. LiHMDS gave a clean profile and good assay yield for the carbonate opening, but the
resulting lithium carbonate gave a slow conversion in the S Ar reaction and higher impurity
N
levels. The carbonate opening reaction was found to be essentially instantaneous; the NaHMDS
reacting immediately with the cyclic carbonate 50 to produce the open carbonate 51 in typically
9
2-100% assay yield. While the carbonate opening reaction was not particularly temperature
sensitive, it was exothermic with an adiabatic temperature rise of 22 °C. Therefore, the carbonate
opening was conducted at -5 °C. Any excess base resulted in a lower assay yield for the reaction;
believed to be a result of open carbonate 51 decomposition, possibly through elimination of HF.
A base screen for the S Ar reaction revealed that NaHMDS, NaOtBu, and KOtBu were the
N
best options, giving complete conversion of the open carbonate and assay yields of 78-90%. The
use of KOtBu resulted in a slightly higher assay yield and a faster reaction when compared to
NaOtBu. The addition of a small amount of water to the slurry of KOtBu and hydroxyproline 46
was found to have a positive impact on the yield of the S Ar reaction. The exact role of the water
N
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
6
added to the hydroxyproline 46 slurry remains unclear; however, it may serve to reduce the
basicity of the reaction mixture by formation of KOH and/or KOTMS. Reactions devoid of water
had lower yields than those with water deliberately charged.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The main process impurity formed in this reaction was the dimer 54, with the proposed
2
2
pathway shown in Table 3. Throughout the course of development, the parameters impacting
the level of dimer 54 were found to be the addition time of the dianion slurry, the total amount of
water in the slurry preparation, and the S Ar reaction temperature during the slurry charge.
N
These parameters were studied in a multivariate fashion, with the results summarized in Table 3.
Based on these results, the process was conducted with an addition time as short as possible
while maintaining the S Ar reaction temperature and the water content within the targeted range.
N
With control of these reaction parameters, the allylic alcohol 49 was routinely isolated in 69%
yield as the benzhydrylamine salt. Conversion of 49 to 31 was accomplished as described earlier
for the deprotection and methyl ester formation. The overall yield of allylic alcohol 31 from
quinoxaline 32 was 59% for the dihydroxylation route, a significant improvement over the 40%
for the epoxidation route.
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Table 3. Proposed Pathway and Multivariate Study on the Formation of Dimer 54
F
F
F F
O
O
N
N
O
N
N
N
F
N
O
F
F
F
O
O
O
N
N
51
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BocN
BocN
HO
O
-
CO2
after
quench
O
O
O
O
BocN
54
52
53
OH
O
Water Amount Addition Time
S
N
Ar Temp.a
(°C)
Dimer 54 b
(HPLC area %)
Entry
Assay Yield
(kg/kg 50)
(h)
1
2
3
4
5
6
7
0.025
0.031
0.036
0.029
0.031
0.036
0.031
0.05
0.05
0.05
0.07
6.5
-10
-10
-10
5
2.5
2.8
71.9%
71.3%
69.7%
65.2%
62.1%
N/A c
4.9
6.3
-15
0
7.6
5.4
13.0
7.3
0
13.9
41.1%
(a) Temperature of the S
(c) Not measured.
N
Ar reaction during the addition of the slurry of 46. (b) Level in the reaction completion sample.
Determining the Origin of Selectivity in Allylic Alcohol Formation
To determine why the carbonate opening leads to higher trans selectivity than the epoxide
opening, a computational study was undertaken. To simplify the calculation, the epoxide was
modeled using the chloride substrate 56 shown in Scheme 12, consistent with the cyclic
carbonate 50. Additionally, counter-ion effects were ignored (and the counter ions not included
in the calculation). The Supporting Information details the methods used for the computational
analysis.
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 12: Substrates Investigated in the Computational Study
N
N
Cl
N
N
Cl
N
N
Cl
O-
3
3
3
1
4
2
4
4
O
2
O-
O
O
O
2
F
F
F
F
1
F F
1
O
O
O
50
55
51
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
Cl
N
N
Cl
N
N
Cl
3
3
3
1
4
2
4
4
2
O-
N
O
2
F
F
F
F
F
F
1
1
O-
56
57
58
Transition states leading to cis and trans products were located for both substrates. In the
carbonate system, the transition state leading to the cis product was found to be 4.4 kcal/mol
higher in energy than the transition state leading to the trans product. This would correspond to a
>4000:1 selectivity for the trans isomer, which is larger than observed experimentally (Table 4,
entry 2), but within computational error.23 In the epoxide system, the transition state leading to
the cis isomer was found to be only 0.4 kcal/mol higher in energy than the trans isomer,
corresponding to a 2.3:1 selectivity for the trans. The difference between the calculated and
experimental selectivity observed with the epoxide (Table 4, entry 1) can be rationalized by the
increased steric bulk in the region of interest for epoxide 48 compared to the model epoxide 56.
Table 4. Calculated versus Observed Levels of Olefin Isomers
Entry
Substrate
Calculated trans:cis Observed trans:cis
1
2
Epoxide 56
2.3:1
12.9:1
200:1
Cyclic Carbonate 50
4000:1
The transition states identified for both substrates show significant E2 character, with
shortening of the C2-C3 bonds, hybridization of the carbon atoms in the forming olefin, and the
lengthening of the carbonate C1-O bond (Figure 2 and Figure 3). The degree of hybridization in
the carbon being deprotonated suggests the transition state is early, and that the elimination itself
is asynchronous, with significant negative charge accumulation on the deprotonated carbon.
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Additionally, the neighboring C3-C4 bond is also shortened, evidence of negative
hyperconjugation between the developing anion and the σ* of the antiperiplanar C-F bond. This
stabilizing interaction is observed in both the cis and trans transition states for each substrate,
and the bond lengths suggests a similar amount of partial double bond character along the
forming olefin and C3-C4 bond.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
syn-pentane steric interaction
1.47
N
1.48
1
.48
Cl
1.48
N
1
.50
Cl
3
1
2
4
3
N
4
2
O
O
N
O
F F
1.42
O
F F
1.41
1
O
O
1
.50
carbonate-TS trans
G‡ = 0 kcal/mol
carbonate-TS cis
G‡rel= 4.4 kcal/mol
rel
Figure 2. Carbonate transition states with steric interactions highlighted. Left: transition state leading to the trans isomer. Right:
transition state leading to the cis isomer. Bottom: Bond lengths (reported in angstroms) for both transition states. The base has
been omitted for clarity.
The largest contributor to the energy difference between the cis and trans isomers for the
carbonate substrate appears to be a syn-pentane-like steric interaction between one of the fluorine
atoms and the terminal carbon of the carbonate (Figure 2). The orbital overlap requirements for
the elimination and the stabilizing interactions with the C-F bond forces the pro-cis carbonate
transition state into a conformation that exhibits a syn-pentane like interaction between C1 and
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
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one of fluorine atoms on C4, resulting in increased steric interaction. In the pro-trans case, the
interaction is lessened to the fluorine experiencing steric interaction with a hydrogen atom. The
difference in steric interaction alone is enough to account for the 4.4 kcal/mol energy difference.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
steric interaction
1.49
1.47
1.49
1.48
N
Cl
N
Cl
3
3
1.46
4
2
4
2
1
N
N
1.43
O
F F
F F
O
.49
1.42
1
1
epoxide-TS trans
G‡rel= 0 kcal/mol
epoxide-TS cis
G‡ = 0.4 kcal/mol

rel
Figure 3. Epoxide transition states. Left: transition state leading to the trans isomer. Right: transition state leading to the cis
isomer. Bottom: Bond lengths (reported in angstroms) for both transition states. The base has been omitted for clarity.
In the epoxide substrate (Figure 3), this type of steric interaction is not observed due to the
bond angles of the epoxide. Instead of the carbon experiencing syn-pentane like interactions, C1
is staggered between the fluorine atoms, avoiding the steric strain. In the pro-cis transition state,
there is some interaction between the epoxide oxygen and one of the fluorine atoms (Figure 3,
right), which explains the transition state being 0.4 kcal/mol higher in energy.
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Strategy for the Synthesis of Macrocycle 3
The key observation of the facile ring closure of the allylic bromide 12 to the macrocycle 3
(Table 2) led to the development of the process to couple allylic alcohol 31 and carbamate 13 to
afford the diol 59, outlined in Scheme 13. The selective conversion of the diol 59 to the allylic
bromide 12 would set the stage for the key ring closure. For a highly functionalized molecule,
special attention would need to be paid to the selectivity of the reactions, impurity formation and
impurity rejection. The impurity profile of macrocycle 3 would need to be well understood as it
is the penultimate intermediate for the synthesis of glecaprevir and any impurities in this stage
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
could impact the final quality of the drug substance.
Scheme 13: Macrocycle 3 Synthetic Route
F
F
F F
HO
N
N
HO
N
N
OH
O O
O
HATU, iPr2EtN
DMA, TMSCl
O
OH
O O
O
OHHN
HN
HCl
0 to 20 °C
O
N
HN
HN
O
O
O
O
13
31
59
F
F
F
F
Br
N
N
N
N
O
O
O
PPh , Br ,
OH
O O
3
2
1
) BnMe N OH
3
CH CN
3
CH CN, H O
3
2
O
0 to 20 °C
O
N
-
10 °C
N
OH
O
HN
O
2) cysteamine HCl
O
O
O
N
H
3) p-Xylene
Formic Acid
H O
2
12
3
77% to 82% yield from 31
As the development of these stages progressed, it was apparent that isolation of the diol 59 or
the allylic bromide 12 would be challenging due to less than ideal physical properties of the
intermediates, and in this regard a telescoped process was targeted. The development of a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
telescoped process then added the requirement that the process streams be compatible with the
next step. From the enabling route, it was known that the crystallization of the macrocycle 3
provided significant purification, and this isolation was targeted as a key control point for
rejection of process impurities.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Development of the Coupling
The best conditions identified for the coupling of allylic alcohol 31 with carbamate 13 utilized
DIPEA as the base, HATU as the acid activation agent, and DMA as the solvent. Under these
conditions the formation of a side product resulting from the acylation of the allylic alcohol was
also observed to the extent of 5%. It was possible to reduce this side reaction via pretreatment of
31 with TMSCl, causing in situ protection of the allylic alcohol. Less than 1% of the side product
was formed when the process was conducted under these improved conditions. After an
extractive workup with toluene and a solvent switch to acetonitrile, the solution of diol 59 was
used directly in the bromination step.
Development of the Bromination
With the diol intermediate 59 in hand, a method was needed to selectively transform the allylic
alcohol to the allylic bromide in the presence of other functionality, including a secondary
alcohol. While a number of conditions are available for this transformation, the selectivity
imparted by the use of phosphonium reagents was quite attractive for further development.24
The bromination reaction was initially developed to isolate a crude solution of 12. Conversion
of 59 to 12 could be affected by bromination using PPh Br in DCM. The reaction mixture
3
2
needed to be quenched into vigorously stirred aqueous NaHCO to prevent bromination of the
3
secondary cyclopentyl alcohol that was observed during extended reaction times and at higher
temperatures. These issues on workup could be solved by telescoping the reaction quench
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directly into the cyclization reaction. This telescoped process was facilitated by changing the
solvent from DCM to the aqueous miscible MeCN.
The bromination reaction was found to be sensitive to water, resulting in lower conversion and
decreased purity. To minimize exposure to moisture, the PPh Br reagent was prepared in situ by
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
addition of Br to a fine suspension of PPh in MeCN. The cleanest bromination reactions used a
2
3
slight excess of Br in the formation of the PPh Br reagent. The resulting PPh Br slurry (1.25
2
3
2
3
2
equiv) was added to the solution of diol 59 at -10 °C. The reaction was exothermic, and the
transfer was conducted at a rate to maintain an internal temperature below 0 °C. The solution of
allylic bromide 12 was used directly in the cyclization reaction.
Development of the Cyclization Reaction
A strong base, such as hydroxide, was necessary for the cyclization of 12 to 3 (Table 5). Under
the reaction conditions, saponification occurred at a faster rate than the cyclization, with
uncyclized bromo-acid intermediate 60 being the last species to cyclize and as such was used for
monitoring for reaction completion. A base screen was conducted (Table 5) and the use of a
quaternary ammonium counterion, such as with Triton® B (BnMe NOH), showed a significantly
3
faster reaction rate when compared to LiOH, NaOH or KOH. Additionally, the use of Triton® B
also minimized the amount of hydrolysis of the allylic bromide to the diol impurity 61. An
attempt to minimize the amount of Triton® B did not show any improvement; combination of 1
equiv of Triton® B with 4 equiv of KOH was slower with higher formation of diol 61 than
Triton® B alone.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Base Screen for the Cyclization of Allylic Bromide 12
F
F
F
F
F F
Br
N
N
N
N
X
N
N
O
O
O
O
OH
O O
OH
O O
Base
O
N
O
O
N
N
OR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
HN
HN
MeCN
water
OMe
OH
O
O
O
O
N
H
12
3
R = H
8 R = Me
60 X = Br
61 X = OH
1
HPLC Area %
Entry
Base
Equiv
Time (h)
1
2
3
86
12
4
18
60
3.5
50
39
64
48
61
3.4
3.5
2.7
1.6
4.2
1
2
3
4
5
Triton® B
KOH
5
5
19
0
0
23
15
34
14
2
13
10
6
NaOH
LiOH
5
21.5
21.5
17.5
5
8
Triton® B, KOH
1, 4
27
14
The ideal amount of 4.5 equiv of Triton® B was necessary as the base for cyclization,
saponification of the ester, and to quench the bromination reagent and by-products. The reaction
was run at 0 °C for 12 hours then warmed to 20 °C until the bromo-acid intermediate 60 was
consumed. The cyclization was performed at these temperatures to minimize the formation of the
diol impurity 61 which was formed at higher levels at warmer temperatures, while also
minimizing cycle time by ensuring complete conversion at the warmer temperature.
Cyclization of Diastereomeric Impurities.
In order to develop a control strategy that ensured the chiral purity of macrocycle 3, impurity
fate and purge studies were conducted with the potential stereoisomers of the chiral components
38, 40, and 46. These fate and purge studies revealed that the potential stereoisomers were
completely cleared in the isolation of macrocycle 3, or in earlier isolated intermediates (13, 49,
or 31). Several of the potential stereoisomers of allylic bromide 12 were carried through the
cyclization reaction, as outlined in Scheme 14 for the enantiomer of hydroxy proline 46. The
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allylic bromide stereoisomer derived from ent 46 resulted in only 36% cyclization to the
corresponding macrocycle bis-epi 3, with hydrolysis of the allylic bromide being a major side
reaction forming 30% of bis-epi 61. A low level of cyclization (49%) was also observed for the
allylic bromide stereoisomer derived from the carboxylate epimer of hydroxy proline 46, with
hydrolysis of the allylic bromide occurring to the extent of 42%. These results were somewhat
surprising given that allylic bromide 12 undergoes 86% cyclization to macrocycle 3, as shown in
entry 1 of Table 5. Based on these observations, the conformation of allylic bromide 12
facilitates cyclization to the macrocycle at a significantly faster rate than hydrolysis, in contrast
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to the other allylic bromide stereoisomers.25
Scheme 14: Cyclization of an Allylic Bromide Stereoisomer
F
F
F
F
N
N
F F
Br
N
N
HO
N
N
(
R)
O
O
OH
O
3
8
O
OH
O
(R)
OH
OH
BocN
O
O
O
O
O
N
OH
O
N
O
N
O
CO2H
CO2H
HN
HN
O
O
OMe
OH
H2N
ent 46
N
H
O
O
40
bis-epi 12
bis-epi 3
bis-epi 61
36%
30%
Reactive Quench of Allylic Bromides
At the end of the reaction, a small amount of bromo-acid 60 remains, along with several other
impurities that contain the allylic bromide functional group. As part of the mutagenic impurity
control strategy, based on ICH M7 guidance, the allylic bromides were assigned as class 3 unless
an Ames test had been performed, and then as Class 2 if the Ames test was positive or Class 5 if
the Ames test was negative.26 In order to develop a control strategy for all the allylic bromides
potentially present, a quench using 2-mercaptoethylamine (cysteamine) was used to react with
the entire class as shown in Scheme 15 for two allylic bromides. Displacement of the allyl
bromide functional group with the thiol results in daughter impurities classified as non-
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