Process Development and Synthesis of Birinapant: Large Scale Preparation and Acid-Mediated Dimerization of the Key Indole Intermediate

Article abstract of DOI:10.1021/acs.oprd.5b00390

Birinapant/TL32711 (1) is a novel bivalent antagonist of the inhibitor of apoptosis (IAP) family of proteins which is currently in clinical development for the treatment of cancer and hepatitis B virus (HBV) infection. In this report, we present a detaile

Full text of DOI:10.1021/acs.oprd.5b00390

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Full Paper
Process Development and Synthesis of Birinapant – Large Scale
Preparation and Acid-mediated Dimerization of the Key Indole Intermediate
Yijun Deng, Qiuzhe Xie, Matthew G. LaPorte, Anna T. A. Chasnoff, Mark A. Mortensen, Debasis Patra,
Seth A. Putrelo, Robert S. Antonovich, Hong Cao, Jun Yan, Arthur J. Cooper, Susan R. Rippin, Matthew
D. Alexander, Pavan T. Kumar, Mukta S. Hendi, Yu-Hua Lee, Thomas Haimowitz, and Stephen M. Condon
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00390 • Publication Date (Web): 06 Jan 2016
Downloaded from http://pubs.acs.org on January 7, 2016
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Process Development and Synthesis of Birinapant −
Large Scale Preparation and Acidꢀmediated
Dimerization of the Key Indole Intermediate
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Yijun Deng,§* Qiuzhe Xie, ‡ Matthew G. LaPorte,§ Anna T. A. Chasnoff, ‡ Mark A. Mortensen, ‡
Debasis Patra, ⊥ Seth A. Putrelo, ⊥ Robert S. Antonovich, ‡ Hong Cao,‡ Jun Yan, ‡
Arthur J. Cooper, ǁ Susan R. Rippin,§ Matthew D. Alexander,§ Pavan Tirunahari Kumar,§
Mukta S. Hendi,§ YuꢀHua Lee,§ Thomas Haimowitz,§ and Stephen M. Condon§*
§TetraLogic Pharmaceuticals Corporation, 343 Phoenixville Pike, Malvern, PA 19355, United
States
‡Albany Molecular Research, Inc., 21 Corporate Circle, Albany, NY 12212, United States
⊥Albany Molecular Research, Inc., 7001 Performance Drive, North Syracuse, NY 13212, United
States
ǁRicerca Biosciences, 7528 Auburn Road, Concord, OH 44077, United States
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Table of Contents Graphic:
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OAc
OH
F
F
O
H
N
N
N
N
N
Me
Me
N
H
Cbz
Cbz
F
OH
NH
NH
O
O
NH
NH
9
+
N
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Cbz
H
N
CO2H
NH
N
H
O
F
F
OAc
OH
Birinapant/TL32711 (1)
9
12
21
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KEYWORDS: birinapant, Smacꢀmimetic, peptide, biindole, hydroxyproline
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ABSTRACT
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Birinapant/TL32711 (1) is a novel bivalent antagonist of the inhibitor of apoptosis (IAP) family
of proteins which is currently in clinical development for the treatment of cancer and hepatitis B
virus (HBV) infection. In this report, we present a detailed description of the 1 drug substance
synthesis used to support our ongoing clinical studies. Key transformations in this process
included the development of a scalable, highꢀyielding route to acyl indole 14 as well as a twoꢀ
step dimerization/oxidation of indole 8 that afforded biindole 21 in excellent yield and purity
(70% yield, 2 steps; >95 area% purity by HPLC analysis). In addition, partial defluorination of
21 was observed following hydrogenꢀmediated benzyloxycarbonyl (Cbz) protective group
removal which was obviated by the use of HBr/HOAc for this transformation. The use of
commerciallyꢀavailable amino acid derivatives afforded related impurities which proved difficult
to purge in subsequent steps. Thus, defining the impurity specification for these reagents was
critical to providing 1 drug substance of >99 area% chemical purity. Using this process, we have
successfully prepared 1 drug substance multiple times on >500 gꢀscale in support of our clinical
development program.
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INTRODUCTION
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Apoptosis, or programmed cell death, is required for normal cellular function, tissue
development and homeostasis.1 The inhibitor of apoptosis, or IAP, family of proteins include the
X chromosomeꢀlinked inhibitor of apoptosis protein (XIAP),2 cellular IAP 1 (cIAP1) and
cIAP2,3 and melanoma IAP (MLꢀIAP),4 amongst others.5 Many of these proteins have wellꢀ
described roles in the suppression of apoptosis under normal physiological conditions.6
Inhibition of apoptosis might also contribute to the initiation and propagation of many cancers
and further the development of drug resistance.7 Inhibiting the IAPs therefore is an attractive
strategy for anticancer drug development.8
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The second mitochondriaꢀderived activator of caspases (Smac/DIABLO) is the endogenous
antagonist of IAP proteins.9 Structural and biological studies have shown that the interaction
between the Nꢀterminal tetrapeptide of Smac, AVPI, and IAPs is mediated through a shallow
groove present on certain baculovirus IAP repeat (BIR) domains of the IAPs. Several small
molecule mimetics of AVPI, termed IAP antagonists, are being advanced in clinical trials for the
treatment of cancer.10 Birinapant/TL32711 (1, Figure 1) is a novel bivalent antagonist of the
IAPs which is currently in clinical trials for the treatment of cancer and HBV infection.11,12 Here
we report the development of a synthetic process for the kgꢀscale preparation of 1 drug substance
to support earlyꢀphase clinical development.
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Figure 1. The chemical structure and ORTEP diagram of 1.
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RESULTS AND DISCUSSION
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Retrosynthetic Analysis of 1. We envisioned that the synthesis of 1 would rely on the parallel
assembly of the two dipeptide chains from a central biindoleꢀcontaining template represented by
A (Figure 2). To prepare the biindole nucleus, further retrosynthetic analysis suggested the
acidꢀcatalyzed dimerization of monomer unit B followed by oxidation.13 Thus, the preparation of
monomer B, including the appropriate selection of heteroatom protective groups (PG1 and PG2),
became the initial focus of our development efforts.
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OPG2
F
OPG2
OH
F
F
N
O
H
N
PG1
N
N
N
N
NH
PG1
PG1
Me
Me
N
H
NH
NH
NH
NH
O
O
+
H
N
NH
N
H
PG1
N
O
F
F
OPG2
OH
F
OPG2
Birinapant (1)
A
B
Figure 2. Retrosynthetic analysis of 1.
The Discovery Route to Indole 8. During the discovery phase of the program, we relied upon
a synthetic route which allowed for the construction of multiple series of 2ꢀarylalkyl or 2ꢀ
heteroarylalkyl pyrrolidines.14 For the eventual discovery of the biindole series of IAP
antagonists represented by 1, we identified indole 8 as a key synthetic intermediate (Scheme 1).
Towards this end, CbzꢀHypꢀOMe (2) was converted to ester 3 and then to 4 following standard
procedures.15 Alkylation of 5 with mesylate 4 afforded the desired acetanilide (6). Treatment of
6 with palladium acetate effected indole formation; then, removal of the pendent acetyl group
provided indole 7 as an oil.16 Inversion of the secondary alcohol was accomplished in three steps
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to yield the partiallyꢀprotected monomer unit (8). Although this synthetic route provided
sufficient quantities of 8 to support the early drug discovery program, the low overall yield (ca.
10%) and reliance on several chromatographic purifications made this process unsuitable for
further development.
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Scheme 1. FirstꢀGeneration Route to Indole 8
Br
F
OH
OTBS
OTBS
HN
1. TBSCl, imid., DCM
2. LiBH4, THF
5. DIBAL/DCM
6. MsCl, TEA, DCM
O
7.
5
N
N
N
Cbz
Cbz
Cbz
3. Swern [O]
4. HWE homologation
CO2Me
NaH, THF
CO2Et
OMs
Cbz-Hyp-OH (2)
3
4
10. TBAF, THF
OH
OTBS
OTBS
F
F
8. Pd(OAc)4, n-Bu4NCl,
11. p-NO2-C6H4-CO2H,
NaHCO3, K2CO3
DIAD, Ph3P, THF
N
N
N
Br
F
Cbz
Cbz
Cbz
12. NaOH, THF
9. NaOH/MeOH
NH
NH
N
O
6
7
8
Process Development of Indole 8 Synthesis. The second generation retrosynthetic analysis of
8 is shown in Figure 3. We envisioned that 8 could be accessed via the reduction of ketone C
which, in turn, would be prepared via the coupling of Oꢀprotected CbzꢀHypꢀOH (D) with an
organometallic species derived from 6Fꢀindole (E). Macor et al. have described the C3ꢀ
acylation of indoles using the acid chloride derived from CbzꢀProꢀOH and indoleꢀderived
Grignard reagents in benzene17 and this strategy has been exploited to prepare the antiꢀmigraine
agent eletriptan.18 From literature reports, we recognized that the choice of the organometallic
indole species E and solvent might have a profound effect on the ratio of C3ꢀ versus N1ꢀ
acylation products.19 For our program, we would also need to determine at which stage to
perform the inversion of the secondary alcohol of CbzꢀHypꢀOH.
F
OPG3
OPG3
OH
F
F
N
N
N
+
Cbz
Cbz
Cbz
N
CO2H
NH
NH
Metal
O
8
C
D
E
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Figure 3. An alternative retrosynthetic analysis of indole 8.
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As the tertꢀbutyldimethylsilyl (TBS) group was employed in the first generation synthesis,
commerciallyꢀavailable CbzꢀHypꢀOH (9, ≥98% purity by HPLC) was reacted with TBSCl
which, following workꢀup, afforded CbzꢀHyp(TBS)ꢀOH (10, Scheme 2).20 Although
dichloromethane solvent could be employed in the acid chloride formation, subsequent reaction
with indoleꢀderived Grignard reagents is reported to require an aromatic solvent.19,21 Thus,
toluene was employed for the acid chloride formation. A toluene solution of 10 was treated with
oxalyl chloride and DMF (cat.) at 25 °C to generate CbzꢀHyp(TBS)ꢀCl (11). The reaction
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mixture was partiallyꢀconcentrated at 40ꢀ45 °C under reduced pressure to remove HCl and
excess oxalyl chloride. At temperatures >50 °C, we observed lower yields of 11 which we
attributed to the loss of the TBS group and subsequent polymerization of the formed CbzꢀHypꢀCl
(not shown).
Scheme 2. Synthesis of CbzꢀHyp(TBS)ꢀCl in Toluene Solution
OH
OTBS
OTBS
TBSCl, TEA
DBU, DMF
oxalyl chloride
Cbz
toluene, DMF
N
N
N
Cbz
Cbz
OH
OH
Cl
O
O
O
9
10
11
In advance of the key indole acylation, a solution of 6Fꢀindole (12) in toluene was treated with
3.0 M EtMgBr in diethyl ether to form a 6Fꢀindoylmagnesium bromide solution (Scheme 3).
The choice of solvent for the acylation reaction proved to be critical. The use of toluene as a
single solvent resulted in agglomeration when toluene solutions of 11 and 12·MgBr (2 equivs)
were combined. A mixedꢀsolvent screen consisting of diethyl ether, THF, MTBE, toluene, and
chlorobenzene identified chlorobenzene as the preferred coꢀsolvent (Supporting Information,
Table S4). Under this condition, the desired acyl indole (13) together with 5ꢀ10% 14 formed
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without any precipitation throughout the addition process. To obviate the separation of 13 and
14, the homogeneous reaction mixture was further diluted with acetic acid (HOAc)/MeOH and
treated with methanesulfonic acid (MSA) resulting in the precipitation of 14 directly from the
reaction mixture. On kgꢀscale, the fourꢀstep transformation of 9 was accomplished without the
isolation of any intermediates to afford 14 in ca. 73% overall yield (based on input 9; >98 area%
by HPLC).
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Scheme 3. Oneꢀpot Acylation of 6ꢀFluoroindolylmagnesium Bromide
OTBS
OH
F
F
1. EtMgBr/Et2O,
toluene, chlorobenzene
MsOH, HOAc, MeOH
N
N
Cbz
Cbz
N
F
2. 11, toluene
(64-73% from 9)
H
NH
NH
O
O
12
13
14
Inversion of the secondary alcohol under the Mitsunobu condition22 gave paraꢀnitrobenzoate
15 which, without isolation, was saponified to afford ketoꢀalcohol 16 as an easily filterable
whiteꢀcolored solid in ca. 70ꢀ80% yield (from 14, >98 area% by HPLC, Scheme 4). Importantly,
HPLC analysis indicated no formation of the nonꢀinverted nitrobenzoate which was prepared by
independent synthesis (not shown). In addition, isolated 16 was absent of any unreacted 14
further confirming the efficiency of this twoꢀstep transformation. Carbonyl group reduction of
16 was accomplished using 2.0 M LiBH4 (2.5 equivalents) and MSA (1.8 equivalents) in THF at
room temperature to give 8 as an isolable solid in ca. 70ꢀ80% yield (>97 area% by HPLC).
Scheme 4. Mitsunobu Inversion of the Secondary Alcohol and Ketone Reduction
O
O2N
OH
O
OH
OH
F
F
F
F
p-NO2-C6H4-CO2H,
DIAD, Ph3P, THF, DMF
LiBH4, MSA,
THF
NaOH, MeOH,
THF, water
N
N
N
N
Cbz
Cbz
Cbz
Cbz
(70-83%)
(85-90%)
(66-81%)
NH
NH
NH
NH
O
O
O
15
16
8
14
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Alternative Synthesis of Indole 8. In the course of this investigation, we observed that ketone
8 was reduced using NaBH4/BF3 etherate to afford 17 after TBS group removal (1 M TBAF in
THF, Scheme 5). Inversion of the secondary alcohol was accomplished using standard
conditions and the resultant nitrobenzoate (18) was saponified to give 8. Although these
reactions proceeded satisfactorily on gramꢀscale, a significant disadvantage to this route was that
intermediates 17 and 18 were isolated as oils. As intermediates 14ꢀ16 (Schemes 3 and 4) were
recrystallizable solids, this alternative route was not pursued further.
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Scheme 5. An Alternative Route to Indole 8
O
OTBS
F
OH
OH
F
F
O2N
O
NaBH4,
p-NO2-C6H4-CO2H,
F
NaOH, MeOH,
THF, water
BF3 etherate
DIAD, Ph3P, THF, DMF
N
N
N
Cbz
Cbz
Cbz
N
then
TBAF/THF
NH
Cbz
NH
NH
O
NH
13
17
18
8
TFAꢀMediated Indole Dimerization. The dimerization of tryptophan derivatives and other
indoles in neat trifluoroacetic acid (TFA) has been reported.13 Unfortunately, the TFAꢀmediated
dimerization of 8 afforded multiple unidentified products by HPLC analysis possibly due to
instability of the intermediate indoylindolines (not shown). We reasoned that the free alcohol
group might be contributing to this poor outcome so we formed the acetyl ester (19) using acetic
anhydride and Nꢀmethylmorpholine (NMM)/DMAP in THF in quantitative yield with only
minimal (ca. 0.5%) indole Nꢀacetylation (Scheme 6).23 To facilitate transfer into the
dimerization reaction, 19 was provided in DCM solution. In the reaction, 19 in DCM (3
volumes) was added to preꢀcooled (ꢀ10 °C) TFA (6 volumes). Higher DCM/TFA ratios resulted
in substantially reduced reaction rates. Under these optimized conditions, ca. 85% consumption
of 19 (HPLC analysis) was observed at 3 h postꢀaddition. In an effort to consume the remaining
19, DCM was removed under reduced pressure thus allowing the reaction to proceed to >95%
completion. The acidic reaction mixture was carefully quenched by reverse addition into
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aqueous K2CO3 and the indoylindoline diastereomers (20) were extracted into isopropylacetate
(IPAc). In advance of the DDQ oxidation, the IPAc was replaced with MeOH. Oxidation of 20
with DDQ in MeOH afforded 21 which precipitated while the DDQ byꢀproduct remained in
solution. Following isolation, crude 21 was recrystallized from DMF/MeOH to provide 21 as an
offꢀwhite solid in ca. 70% yield (from 8, >98 area% by HPLC).
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Scheme 6. TFAꢀMediated Dimerization of Indole 19 to Form Biindole Core Template
OAc
OAc
F
F
N
N
N
N
H
H
Cbz
TFA/DCM
Cbz
DDQ, MeOH
OH
OAc
F
F
Ac2O, NMM,
DMAP, THF
NH
NH
NH
NH
N
N
Cbz
Cbz
(64-72%
NH
NH
from 8)
Cbz
Cbz
8
19
F
F
OAc
OAc
21
20
Stereochemical Purity of Biindole 21. Biindole 21 contains four chiral centers giving rise to
16 possible stereoisomers which can be further reduced to six racemic pairs after accounting for
the C2ꢀsymmetry of the molecule (Figure 4A). To confirm the stereochemical purity of 21, we
prepared each of these stereoisomers using modifications of known methods (Supporting
Information). As shown in Figure 4B, 21 was resolved from each of the other stereoisomers
using reversedꢀphase (C18) HPLC analysis when prepared as an artificial mixture of
stereoisomers. Chromatographic analysis of an authentic sample of 21 demonstrated its
chemical purity and confirmed the sterochemical purity of the indole congener (8, Figure 4C).
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Figure 4. The stereochemical purity of 21. A. The six possible stereoisomers of 21; B.
HPLC chromatogram of an artificial mixture of 21ꢀ26 demonstrating the separation of 21
from the other stereoisomers; and, C. HPLC chromatogram of authentic 21.
The First Peptide Bond Formation. Having secured a robust, kgꢀscale synthesis of 21, we
turned our attention to the sequential coupling with the requisite amino acid derivatives.
Removal of the two Cbz groups was routinely achieved on multigramꢀscale using 10% Pd/C in
MeOH under hydrogen atmosphere (50 psi) using a Parr Shakerꢀtype hydrogenation apparatus
which afforded 27 in quantitative yield (Scheme 7). Application of these conditions on >100 gꢀ
scale, however, resulted in ca. 1ꢀ1.5% of the monoꢀfluorinated derivative 28 which was
confirmed by independent synthesis.24 More importantly, we were unable to remove 28 by
recrystallization of 27 or at a later stage in the synthesis. In order to identify hydrogenation
conditions that either reduced or eliminated formation of 28, we performed a catalyst screen
using the ENDEAVOR® catalyst screening system (Argonaut Technologies, Foster City, CA;
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Supporting Information, Tables S5). Despite identifying promising Pd catalysts, i.e., Escat
1931 (Strem Chemicals), we remained concerned about the generation of this difficultꢀtoꢀpurge
impurity (28) on subsequent scaleꢀup of these conditions. Thus, we opted to explore the use of
HBr/HOAc for the Cbz group removal. In the event, HBr (33 wt% in HOAc) was added to 21 in
DCM at ambient temperature. After approximately 24 h, the reaction mixture was quenched
with water and extracted with methyl tertꢀbutyl ether (MTBE) to remove benzyl bromide. The
productꢀcontaining aqueous phase was made basic (pH 8.0±0.2) with 2 N NaOH and 25 was
extracted into 2ꢀMeTHF followed by a solvent exchange to MeOH which allowed 27 to be
isolated as an offꢀwhite solid (93% yield, >98 area% by HPLC). Under these new conditions, we
were unable to detect 28 in either the crude reaction mixture or isolated 27.
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Scheme 7. Defluorination under Hydrogenation Conditions
OAc
OAc
OAc
F
F
F
N
N
HN
HN
HN
HN
Cbz
Cbz
NH
NH
NH
NH
NH
A. H2, Pd/C, MeOH
+
or
NH
B. HBr, HOAc
(B: 89-93%)
F
F
H
OAc
OAc
OAc
21
27
28
From the outset, we selected HATU as the reagent of choice owing to its ability to couple
stericallyꢀhindered and hydrophobic amino acid derivatives with little or no epimerization.25,26
Reaction of 27 with 2.4 equivalents of BocꢀAbuꢀOH and excess HATU/NMM in 1ꢀmethylꢀ2ꢀ
pyrrolidinone (NMP) gave 29 in excellent yield (Scheme 8). As expected, under these
conditions we observed no epimerization of the Abu stereocenter by HPLC analysis using an
authentic monoꢀDꢀAbuꢀ29 standard (not shown). However, ca. 5% of the desꢀmethyl derivative
(30) was observed in the reaction mixture. We suspected that the formation of 30 came from the
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BocꢀAbuꢀOH which was likely contaminated with BocꢀAlaꢀOH. To confirm this hypothesis, we
prepared an authentic sample of 30 (Supporting Information). During an independent
preparation of 29 using a second lot of BocꢀAbuꢀOH, we observed ca. 2% formation of Abuꢀ
insertion adducts 31 and 32 generated by the coupling of 27 with BocꢀAbuꢀAbuꢀOH dipeptide.
Analysis of several lots of commerciallyꢀavailable BocꢀAbuꢀOH revealed various levels of
contamination with HꢀAbuꢀOH, BocꢀAlaꢀOH, and/or BocꢀAbuꢀAbuꢀOH. As such, commercial
lots of BocꢀAbuꢀOH which did not achieve the desired purity specification were recrystallized
(10:1.5 nꢀheptane/MTBE) prior to use. Notably, we suspect that the formation of 32 arose via Cꢀ
terminal epimerization of HATUꢀactivated BocꢀAbuꢀAbuꢀOH intermediate.27
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Scheme 8. ByꢀProducts 30, 31 and 32 Formed by Contaminated BocꢀAbuꢀOH Reagent
OAc
OAc
OAc
F
F
F
Boc
Boc
Boc
Boc
HN
HN
N
N
N
N
N
H
N
H
Boc-Abu-OH, HATU,
NMM, NMP
NH
NH
O
O
NH
O
O
NH
NH
+
NH
H
N
H
N
F
F
F
OAc
OAc
OAc
27
29
30
OAc
OAc
F
F
O
O
H
H
N
N
N
N
N
O
Boc
Boc
N
H
N
H
O
O
NH
NH
NH
NH
O
N
H
N
H
N
Boc
Boc
F
F
OAc
OAc
31
32
Purification of Diamine 33. The isolation of 29 was problematic as the solid was a very fine
powder and filtration was difficult. Our attempts to generate crystalline 29 with improved
filtration characteristics were unsuccessful. Thus we opted to advance 29 into the next reaction
without isolation. Following BocꢀAbuꢀOH coupling to 27 (Scheme 8), the reaction mixture was
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diluted with MTBE/EtOAc and subjected to aqueous workup. The MTBE/EtOAc extracts were
concentrated, dissolved in DCM, and used directly in the TFAꢀmediated Boc group removal
(Scheme 9). Gratifyingly, 33 was isolated as an easilyꢀfilterable solid in high purity (>97 area%
by HPLC) after workꢀup. Additionally, recrystallization of 33 effectively purged the Abuꢀ
insertion products derived from 31 and 32.
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Scheme 9. Synthesis of Key Intermediate 33
OAc
OAc
F
F
Boc
Boc
N
N
N
N
N
H
H2N
H2N
TFA, DCM
O
O
NH
NH
O
O
NH
NH
(86-96%
from 27)
H
N
F
F
OAc
OAc
29
33
The Second Peptide Bond Formation. The successful coupling of 33 with BocꢀN(Me)Alaꢀ
OH under HATU/NMM conditions afforded 34 contaminated with 1ꢀ1.5% of desꢀmethyl species
35 (Scheme 10). As before, the genesis of 35 could be traced to contamination of commerciallyꢀ
available BocꢀN(Me)AlaꢀOH with BocꢀAlaꢀOH; recrystallization of commercial lots of Bocꢀ
N(Me)AlaꢀOH (15:2 nꢀheptane/MTBE) afforded high purity reagent thus eliminating the
formation of 35.
Fullyꢀprotected birinapant 34 was isolated as a white solid after
recrystallization from MTBE/nꢀheptane.
Scheme 10. Synthesis of Penultimate 34 and DesꢀMethyl Byꢀproduct 35
OAc
OAc
OAc
F
F
F
Me
N
O
Me
N
O
N
N
N
N
N
H2N
H2N
Boc
Boc
N
H
Boc
+
N
H
Boc-N(Me)Ala-OH,
HATU, NMM, NMP
O
O
NH
NH
O
O
NH
NH
O
O
NH
NH
(87-96%)
H
N
H
N
Boc
N
N
N
H
Me
O
O
F
F
F
OAc
OAc
OAc
33
34
35
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Synthesis and Isolation of 1. The final removal of the Boc and acetyl protective groups was
accomplished in twoꢀsteps without isolation of the partiallyꢀprotected intermediate (36, Scheme
11). Fullyꢀprotected 34 was treated with TFA in DCM to afford diamine 36. Following
neutralization and extraction (2ꢀMeTHF), the solvent was replaced with MeOH and then
combined with 1 N NaOH to effect removal of the two acetyl groups. Distillation of MeOH
allowed for the extraction of crude 1 into 2ꢀMeTHF from the aqueous solution. The organic
extracts were concentrated to dryness and the residue was recrystallized in IPA/EtOAc/water to
provide 1 as a crystalline, offꢀwhiteꢀcolored solid.
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Scheme 11. The Synthesis of Birinapant/TL32711 (1)
OAc
OAc
F
F
Me
N
O
O
H
N
N
N
N
N
Boc
Boc
Me
Me
N
H
N
H
O
O
NH TFA, DCM
NH
O
O
NH
NH
H
N
H
N
N
N
H
Me
O
O
F
F
OAc
OAc
34
36
OH
F
O
H
N
N
Me
Me
N
H
O
NH
NH
aq. NaOH, MeOH
(92-94% from 34)
O
H
N
N
N
H
O
F
OH
1
CONCLUSIONS
We have developed a scalable process to synthesize 1 drug substance for clinical use. A major
improvement was the synthesis of intermediate 8 through acid chloride/indoylmagnesium
bromide coupling which improved the yield from ca. 10% to ca. 50% from commerciallyꢀ
available reagents. Unexpectedly, we observed significant levels of defluorination during the
hydrogenation of 21 which mandated an alternative method for Cbz group removal (i.e.,
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HBr/HOAc). This program also revealed the chemical purity of the amino acid derivatives, Bocꢀ
AbuꢀOH and BocꢀN(Me)AlaꢀOH, as critical quality attributes for this process. Finally, all
chromatographic purifications were removed from the entire synthesis and crystalline 1 was
conveniently isolated by filtration. This new synthetic process has allowed multiple >500 g lots
of 1 to be produced for use in clinical trials.
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EXPERIMENTAL SECTION
1
General. H and 13C NMR spectra were obtained at 300 and 75 MHz, respectively, using a
Varian L600 spectrometer using tetramethylsilane as the internal standard. For all prolineꢀ
containing analogs where two sets of resonances are observed owing to the presence of
conformational isomers, the signal set for the major conformational isomer is reported. Reactions
were routinely performed under a nitrogen atmosphere using standard glassware and high purity,
commercialꢀgrade solvents. Amino acid derivatives were purchased from Bachem, Fluka,
ChemꢀImpex or other reputable suppliers and used without further purification unless stated
otherwise. Unless indicated, amino acids and derivatives have the
Lꢀconfiguration. LC/MS
analysis was performed on a ThermoꢀFisher MSQ Plus instrument with a Gemini 5 ꢀ C6ꢀPhenyl
110 Å column (50 × 4.60 mm) using standard gradient conditions (A: water containing 0.1%
HOAc v/v; B: acetonitrile containing 0.1% HOAc v/v); MS data were acquired with ESI,
positive ionization; UV detection at 254 nm. Compound purifications were performed using
flash silica gel chromatography or by preparative highꢀperformance liquid chromatography using
a Varian Prostar system either in normal phase (SiO2, EtOAc/hexane, 250 × 41.4 mm) or
reversedꢀphase (C18, 100 Å, 60 ꢀ, 250 × 41.4 mm). Abbreviations: 2ꢀMeTHF, 2ꢀ
methyltetrahydrofuran; Abu, aminobutyric acid; ACN, acetonitrile; Ac2O, acetic anhydride;
AUC, area under the curve; Boc, tertꢀbutyloxycarbonyl; Cbz or Z, benzyloxycarbonyl; DBU,
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1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene; DCM, dichloromethane; DDQ, 2,3ꢀdicholoroꢀ5,6ꢀdicyanoꢀ
1,4ꢀbenzoquinone; DIAD, diisopropylazodicarboxylate; DIPEA, diisopropylethylamine; DMAP,
4ꢀdimethylaminopyridine; DMF, N,Nꢀdimethylformamide; DMSO, dimethylsulfoxide; EtOAc,
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ethyl
hexafluorophosphate; HOAc, acetic acid; HPLC, highꢀperformance liquid chromatography; Hyp,
ꢀhydroxyproline; IPA, isopropyl alcohol; IPAc, isopropylacetate; MeOH, methanol; MSA,
acetate;
HATU,
Oꢀ(7ꢀazabenzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀtetramethyluronium
L
methanesulfonic acid; MTBE, methyl tertꢀbutyl ether; NMM, 4ꢀmethylmorpholine; NMP, 1ꢀ
methylꢀ2ꢀpyrrolidinone; NMR, nuclear magnetic resonance; TBSꢀCl, tertꢀbutyldimethylsilyl
chloride; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
4Rꢀ(tertꢀButylꢀdimethylꢀsilanyloxy)ꢀpyrrolidineꢀ1,2Sꢀdicarboxylic acid 1ꢀbenzyl ester (10). Zꢀ
HypꢀOH (9, 3.0 kg, 11.3 mol), and N,Nꢀdimethylformamide (DMF, 12.6 L) were charged to a
100 L reactor. The solution was cooled to 10ꢀ15 °C. Triethylamine (3.9 L, 28.3 mol) was added,
followed by addition of 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU, 170 mL, 1.1 mol). To the
solution was added tertꢀbutyldimethylsilyl chloride (TBSꢀCl, 1.9 kg, 12.4 mol) in portions to the
batch maintaining the temperature between 15–25 °C. The batch was stirred at 25 °C for 18 h.
The reaction mixture was cooled to 0±5 °C and quenched with water (15.4 L) while maintaining
the temperature below 20 °C. MTBE (30 L) was added and the mixture was acidified to pH 3ꢀ4
with 4 N HCl (6 L) while maintaining the batch temperature below 20 °C. The organic layer was
separated and washed with water (15 L) and brine (15 L). The organic phase was concentrated to
6 L under reduced pressure at 35ꢀ40 °C. The solution was azeotropicallyꢀdried with toluene (2 ×
5 L) at ≤40 °C to yield 10 (theoretical yield: 4.29 kg, >99 area% by HPLC) in toluene (6 L),
which was stored at 2ꢀ8 °C and used directly in the following reaction. An analytical sample
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was prepared for characterization: 1H NMR (300 MHz, CDCl3): δ 7.36 (br s, 3H), 7.30 (m, 2H),
5.18 (m, 2H), 4.52 (dd, J = 7.5, 15.9 Hz, 1H), 5.43 (m, 1H), 3.40ꢀ3.70 (m, 2H), 2.06ꢀ2.36 (m,
2H), 0.85 (s, 9H), 0.07 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δ 183.1, 181.7, 160.8, 159.5,
141.3, 141.2, 131.4, 133.3, 133.0, 132.8, 132.5, 75.6, 72.5, 72.2, 63.1, 62.6, 60.1, 59.7, 44.8,
43.4, 30.6, 22.9, 0.07 ppm; HRMS(ESI) m/z 380.1891 [(M+H), calcd for C19H30NO5Si:
380.1893].
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2Sꢀ(6ꢀFluoroꢀ1Hꢀindoleꢀ3ꢀcarbonyl)ꢀ4Rꢀhydroxyꢀpyrrolidineꢀ1ꢀcarboxylic acid benzyl ester
1
(14). Step 1: To the solution of ZꢀHyp(OTBS)ꢀOH 10 (4.3 kg, based on H NMR wt% assay,
11.3 mol) in toluene (6 L) was added DMF (4 mL, cat.) followed by the addition of oxalyl
chloride (1.18 L, 13.5 mol) at the rate to maintain the internal temperature below 25 °C. The
batch was stirred at ambient temperature for 2 h. An aliquot (0.2 mL) of the reaction mixture
was removed and added into MeNH2 (2 M in THF, 2 mL). The sample was diluted with MeOH
and analyzed by HPLC to confirm completion of the reaction. The homogeneous reaction
mixture was concentrated under reduced pressure at 40ꢀ45 °C and dried azeotropically with
toluene to provide compound 11 in toluene (12 L) as a pale yellowꢀcolored solution.
Step 2: 6ꢀFluoroindole (12, 3.06 kg, 22.6 mol) was dissolved in anhydrous chlorobenzene (23.1
L) and toluene (15.4 L) and the solution was cooled to ꢀ5ꢀ0 °C. Ethylmagnesium bromide (3 M
solution in diethyl ether, 8.3 L, 24.9 mol) was added at the rate to maintain the temperature
below 5 °C. After stirring for 1 h at 5 °C, 11 in toluene (infra) was added at the rate to maintain
the temperature below 5 °C. The reaction mixture was stirred for 3 h at <5 °C then quenched
with a mixture of water (0.43 L), glacial acetic acid (2.7 L) and MeOH (19 L). The resulting 13ꢀ
containing reaction mixture was used directly in the subsequent reaction.
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Step 3: To the 13ꢀcontaining reaction mixture was charged methanesulfonic acid (MSA, 0.97 L,
14.8 mol) while maintaining the internal temperature below 25 °C. The reaction mixture was
stirred at ambient temperature for 16 h. The solid was collected by filtration and washed with
water (8.5 L) and MTBE (2 × 8.5 L). The isolated solid was slurried in a mixture of water and
MeOH (12% MeOH in water, 72.5 L) and filtered. The wet cake was washed with MTBE (2 ×
8.5 L) and dried under vacuum at 45 °C for 48 h to provided compound 14 as an offꢀwhiteꢀ
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1
colored solid (3.1 kg, 73% yield from 9, >98 area% by HPLC). H NMR (300 MHz, DMSOꢀd6):
δ 12.06 (d, J = 9.3, Hz, 1H), 8.43 (dd, J = 2.1, 9.9 Hz, 1H), 8.16 (m, 1H), 7.35 (br s, 2H), 7.26 (d,
J = 9.9 Hz, 1H), 7.02 (m, 3H), 5.23 (m, 1H), 5.12 (m, 1H), 5.04 (d, J = 6.9 Hz, 1H), 4.91 (q, J =
13.5 Hz, 1H), 4.30 (br s, 1H), 3.54 (m, 1H), 3.46 (br d, J = 10.8 Hz, 1H), 3.33 (br s, 1H), 2.24
(m, 1H), 1.91 (m, 1H) ppm; 13C NMR (75 MHz, DMSOꢀd6), mixture of rotamers: δ 194.4, 193.9,
160.0 (d, JCF = 235.5 Hz), 154.7 (d, JCF = 12.0 Hz), 137.8, 137.4, 137.3, 137.2, 137.2, 135.2,
129.1, 128.50, 128.46, 128.1, 127.9, 127.4, 123.3, 123.1, 123.0, 114.7, 114.5, 111.0, 111.0,
110.8, 110.7, 99.3, 98.9, 69.6, 68.8, 66.5, 66.4, 61.5, 61.1, 56.3, 55.8 ppm; HRMS (ESI), m/z
383.1401 [(M+H), calcd for C21H20FN2O4:383.1396].
2Sꢀ(6ꢀFluoroꢀ1Hꢀindoleꢀ3ꢀcarbonyl)ꢀ4Sꢀ(4ꢀnitroꢀbenzoyloxy)ꢀpyrrolidineꢀ1ꢀcarboxylic
acid
benzyl ester (15). A solution containing 14 (2.9 kg, 7.7 mol), 4ꢀnitrobenzoic acid (1.6 kg, 9.6
mol) and triphenylphosphine (3.6 g, 13.5 mol) in anhydrous THF (49.8 L) and DMF (9.9 L) was
cooled to 0ꢀ5 °C. Methanesulfonic acid (MSA, 199 mL, 3.1 mol) was added, followed by
addition of diisopropylazodicarboxylate (DIAD, 2.8 L, 14.1 mol) at a rate that maintained the
internal temperature below 5 °C. After 1 h at 0ꢀ5 °C, the solution was allowed to warm to
ambient temperature and stirred for 16 h. MeOH (2.9 L) was added. The batch was stirred for 30
min and concentrated under reduced pressure at 45 °C. MeOH (14.7 L) was added and the
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solution was concentrated again to remove residual THF. MeOH was added and the resulting
heterogeneous mixture was stirred at 0 °C for 2 h. The solid was collected by filtration and
washed with MeOH (8 L). The wet cake was dried under vacuum at 35 °C for 16 h to give 15 as
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a pale yellowꢀcolored solid (3.4 kg, 83% yield, >97 area% by HPLC). H NMR (300 MHz, d6ꢀ
DMSO), mixture of rotamers: δ 12.10 (br s, 1H), 8.45 (dd, J = 6.3, 3.0 Hz, 1H), 8.29ꢀ8.20 (m,
3H), 8.02 (dd, J = 2.7, 9.0 Hz, 2H), 7.65ꢀ7.50 (m, 2H), 7.30ꢀ7.42 (m, 2H), 7.26 (dd, J = 2.1, 9.9
Hz, 1H), 7.20ꢀ7.04 (m, 4H), 5.53 (br s, 1H), 5.40 (dd, J = 9.4, 14.4 Hz, 1H), 5.11 (dd, J = 12.6,
21.9 Hz, 1H), 5.04 (s, 1H), 3.97 (m, 1H), 3.71 (d, J = 12.3 Hz, 1H), 2.90 (m, 1H), 2.36 (m, 1H)
ppm; 13C NMR (75 MHz, d6ꢀDMSO), mixture of rotamers: δ 192.9, 192.5, 164.2, 160.0 (d, JCF =
235.9 Hz), 154.5 (d, JCF = 11.8 Hz), 150.9, 137.5 (d, JCF = 2.6 Hz), 137.2 (d, JCF = 12.6 Hz),
135.6, 135.2, 132.7, 132.2 (d, JCF = 9.5 Hz), 131.3, 129.5, 129.4, 129.1,128.7, 128.5, 128.3,
128.2, 127.6, 124.3, 123.1, 113.7, 113.6, 111.0 (d, JCF = 23.5 Hz), 99.7 (d, JCF = 25.7 Hz), 75.3,
74.3, 68.5, 66.8, 66.5, 62.5, 62.2, 53.6, 53.1, 38.7, 37.7, 22.6. HRMS (ESI), m/z 554.1351
[(M+Na), calcd for C28H22FN3O7Na: 554.1359].
2Sꢀ(6ꢀFluoroꢀ1Hꢀindoleꢀ3ꢀcarbonyl)ꢀ4Sꢀhydroxyꢀpyrrolidineꢀ1ꢀcarboxylic acid benzyl ester
(16). To a suspension of 15 (6.5 kg, 12.2 mol) in THF (65 L) and MeOH (19.5 L) was added
50% aq. NaOH (0.97 L) at a rate to maintain the internal temperature below 20 °C. After 1.5 h at
ambient temperature, glacial acetic acid (0.8 L) was added to adjust the pH to 6.5±0.2 and the
reaction mixture was concentrated under reduced pressure at 45 °C. The solvent was exchanged
with MTBE (5 × 11 L), followed by addition of water (65 L). After 1 h at ambient temperature,
the solid was collected by filtration. The wet cake was washed with water and MTBE, dried
under vacuum at 45 °C for 16 h to give 16 as a whiteꢀcolored solid (4.1 kg, 87%, >98 area% by
1
HPLC). H NMR (300 MHz, DMSOꢀd6): δ 12.1 (br s, 1H), 8.38 (d, J = 11.1 Hz, 1H), 8.14 (m,
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1H), 7.40ꢀ7.30 (m, 2H), 7.25 (dd, J = 2.1, 9.9 Hz, 1H), 7.10ꢀ6.95 (m, 4H), 5.16ꢀ4.98 (m, 3H),
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4.95 (dd, J = 13.2, 25.4 Hz, 1H), 4.26 (m, 1H), 3.75 (m, 1H), 3.31 (br s, 1H), 3.22 (m, 1H), 2.60
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(m, 1H), 1.74 (m, 1H) ppm; C NMR (75 MHz, DMSOꢀd6): δ 193.8, 193.3, 160.0 (d, JCF
=
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235.3 Hz) , 154.5 (d, JCF = 14.6 Hz), 137.7, 137.4, 137.2, 135.2 129.1, 128.5, 128.2 (d, JCF = 35.8
Hz), 128.1, 127.5, 123.3, 123.1, 114.5, 114.4, 110.9 (d, JCF = 23.5 Hz), 110.8 (d, JCF = 23.7 Hz),
99.1 (d, JCF = 25.7 Hz), 69.5, 68.7, 66.5, 66.4, 61.5, 61.3, 55.0, 54.7 ppm. HRMS (ESI), m/z
383.1405 [(M+H), calcd for C21H21FN2O4: 383.1407].
2Rꢀ(6ꢀFluoroꢀ1Hꢀindolꢀ3ꢀylmethyl)ꢀ4Sꢀhydroxyꢀpyrrolidineꢀ1ꢀcarboxylic acid benzyl ester (8).
To a suspension of 16 (3.0 kg, 0.22 mol) in anhydrous THF (60 L) at ꢀ10ꢀ0° C was added LiBH4
in THF (2 M, 9.8 L, 19.6 mol) at the rate to maintain the internal temperature below 0 °C. The
resulting mixture was stirred at 0ꢀ5 °C for 30 min followed by addition of MSA (0.92 L, 14.0
mol) at the rate that maintained the batch temperature below 0 °C [Note: vigorous gas evolution
was observed during the addition of MSA]. The reaction mixture was stirred at ambient
temperature for 16 h, and then cooled to 0ꢀ5 °C before a mixture of water and THF (1:5, volume)
was slowly added to quench the reaction. The mixture was concentrated under reduced pressure
at <35°C. The residue was diluted with EtOAc (45 L) and water (30 L). After 15 min, the
organic phase was separated and washed successively with NaHCO3 solution (5 wt%), 2 N HCl,
water and aq. NaHCO3 (5 wt%). The organic phase was concentrated under reduced pressure.
The residue was diluted with MTBE (15 L) and the solvent was exchanged with MTBE. After 1
h, the product was collected by filtration, washed with MTBE and dried under vacuum at 45 °C
1
for 16 h to give 17 as a whiteꢀcolored solid (2.3 kg, 79% yield, >97 area% by HPLC). H NMR
(300 MHz, DMSOꢀd6): ~1:1 mixture of rotamers: δ 10.89, 10.87 (brs, 1H), 7.66 (dd, J = 8.1, 8.1
Hz, 0.5H), 7.50ꢀ7.30 (m, 4.5H), 7.16ꢀ7.04 (m, 3H), 6.84 (dd, J = 8.4, 8.4 Hz, 0.5H), 6.56 (dd, J
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=9.0, 9.0 Hz, 0.5H), 5.13 (br s, 2H), 5.06 (br s, 1H), 4.24 (br s, 1H), 3.98 (m, 1H), 3.55 (dd, J =
4.8, 11.4 Hz, 1H), 3.26 (d, J = 11.4 Hz, 1H), 3.00 (dd, J = 10.5, 13.2 Hz, 2H), 1.79 (m, 2H) ppm;
13C NMR (300 MHz, DMSOꢀd6): mixture of rotamers: δ 159.6 (d, JCF = 231.8, Hz), 159.4 (d, JCF
= 232.4 Hz), 154.9, 138.0, 137.5, 136.8, 136.6, 129.2, 129.1, 128.8, 128.5, 128.2, 125.2, 124.9,
124.5, 124.4, 120.2 (d, JCF = 27.8 Hz), 120.0 (d, JCF = 27.8 Hz), 112.5, 112.4, 107.5 (d, JCF = 24.3
Hz), 107.3 (d, JCF = 23.7 Hz), 97.9 (d, JCF = 25.5 Hz), 69.9, 69.2, 67.2, 66.4, 58.8, 58.1, 56.1,
55.6, 38.4, 37.7, 31.3, 30.1 ppm. HRMS (ESI), m/z 369.1602 [(M+H), calcd for C21H22FN2O3:
369.1603].
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4SꢀAcetoxyꢀ2Rꢀ(6ꢀfluoroꢀ1Hꢀindolꢀ3ꢀylmethyl)ꢀpyrrolidineꢀ1ꢀcarboxylic acid benzyl ester (19).
To a suspension of 8 (2.3 kg, 6.2 mol), 4ꢀmethylmorpholine (NMM, 1.4 L, 12.4 mol) and 4ꢀ
dimethylaminopyridine (DMAP, 76 g, 0.62 mol) in THF (22.8 L) at 15ꢀ20 °C was added acetic
anhydride (0.71 L, 7.5 mol) at a rate that maintained the internal temperature below 20 °C. After
1 h, the reaction mixture was quenched with water (2.3 L). The batch was concentrated under
reduced pressure at 35ꢀ40 °C. The residue was diluted with DCM (23 L) and washed
successively with 2 N HCl, saturated NaHCO3 solution and brine. The organic phase was
concentrated under reduced pressure at 35 °C. The residue containing 19 was diluted with DCM
(3 vol.) and used directly in the next reaction (97.6% yield, >96 area% by HPLC). An analytical
1
sample was prepared: H NMR (300 MHz, DMSOꢀd6), ~1:1 mixture of rotamers: δ 10.92 (br s,
1H), 7.61 (m, 0.5 H), 7.50ꢀ7.30 (m, 5H), 7.02ꢀ7.16 (m, 2.5H), 6.85 (m, 0.5H), 6.58 (m, 0.5H),
5.16 (m, 1H), 5.14 (s, 2H), 4.09 (m, 1H), 3.73 (dd, J = 4.8, 12.2 Hz, 1H), 3.58 (m, 0.5H), 3.40
(m, 1H), 3.28 (d, J = 13.5 Hz, 0.5H), 3.16 (d, J = 12.3 Hz, 0.5H), 2.80 (t, J = 12.3 Hz, 1H), 2.08
13
(s, 3H), 2.05 (m, 1H), 1.70ꢀ1.88 (m, 1.5H) ppm; C NMR (75 MHz, DMSOꢀd6), mixture of
rotamers: δ 170.7, 159.5 (d, JCF = 226.4 Hz), 154.7, 137.7, 137.3, 136.8, 136.7, 129.1, 128.8,
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128.5, 128.3, 125.0, 124.6, 123.0, 119.9, 111.8, 107.7, 107.4, 98.1 (d, JCF = 23.4 Hz), 74.0, 73.2,
67.7, 67.3, 66.6, 58.3, 57.6, 55.5, 53.4, 52.9, 35.2, 34.5, 31.0, 30.0, 25.8, 21.7 ppm. HRMS
(ESI), m/z 411.1760 [(M+H), calcd for C23H24FN2O4: 411.1760].
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4SꢀAcetoxyꢀ2Rꢀ[3'ꢀ(4Sꢀacetoxyꢀ1ꢀbenzyloxycarbonylꢀpyrrolidinꢀ2Rꢀylmethyl)ꢀ6,6'ꢀdifluoroꢀ
1H,1'Hꢀ[2,2']biindolylꢀ3ꢀylmethyl]ꢀpyrrolidineꢀ1ꢀcarboxylic acid benzyl ester (21). The solution
containing 19 (2.5 kg, theoretical yield from previous step, 6.2 mol) in DCM (7.5 L) was charged
to preꢀcooled TFA (15.2 L) at ꢀ10 °C. After 1 h, the batch was concentrated under reduced
pressure to remove DCM at 0ꢀ5 °C and stirred for another 4 h (98% conversion by HPLC). The
cold reaction mixture was transferred into a preꢀcooled (0ꢀ5 °C) mixture of water (64 L), K2CO3
(17.8 kg) and IPAc (38 L). The organic phase was separated and washed with water and
concentrated under reduced pressure below 40 °C. The residue was diluted with IPAc followed
by a solvent exchange to MeOH which was used directly in the next reaction. HRMS (ESI), m/z
821.3372 [(M+H), calcd for C46H47F2N4O8: 821.3362].
To the solution containing the indoylindoline diastereomers (20, 3.1 mol, assuming 100%
conversion from previous step) in MeOH (55 L) at 10ꢀ15 °C was added 2,3ꢀdicholoroꢀ5,6ꢀ
dicyanoꢀ1,4ꢀbenzoquinone (DDQ, 0.7 kg, 2.8 mol) in portions. The resulting slurry was stirred at
ambient temperature for 2 h before the crude product was collected by filtration. The wet cake
was washed with MeOH (2 × 5 L) and dried under vacuum at 40 °C for 16 h to give crude 21
which was recrystallized with DMF (30 L) and MeOH (90 L) to provide 21 as an offꢀwhiteꢀ
1
colored solid (1.8 kg, 72% yield, >98 area% by HPLC). [α]25 = +114.6° (c = 1.0, CHCl3). H
D
NMR (300 MHz, CDCl3): δ 11.29 (br s, 2H), 7.57ꢀ7.36 (m, 14H), 6.90 (app dt, J = 2.1, 9.3 Hz,
2H), 5.39ꢀ5.30 (m, 6H), 4.28 (t, J = 9.0 Hz, 2H), 3.84ꢀ3.73 (m, 4H), 3.66 (d, J = 13.2 Hz, 2H),
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3.40 (dd, J = 12.0, 14.4 Hz, 2H), 2.31 (s, 6H), 2.17 (m, 2H), 2.05 (m, 2H) ppm; C NMR (75
MHz, CDCl3): δ 170.7, 160.4 (d, JCF = 236.1 Hz), 156.3, 137.4 (d, JCF = 13.4 Hz), 136.5, 128.9,
128.6, 128.5, 125.9, 118.7 (d, JCF = 10.6 Hz), 108.8, 108.4 (d, JCF = 8.3 Hz), 98.5 (d, JCF = 25.8
Hz), 74.4, 68.0, 60.1, 53.5, 34.5, 28.9, 21.7 ppm. HRMS (ESI), m/z 819.3252 [(M+H), calcd for
C46H45F2N4O8: 819.3205].
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Acetic acid 5Rꢀ[3'ꢀ(4Sꢀacetoxyꢀpyrrolidinꢀ2Rꢀylmethyl)ꢀ6,6'ꢀdifluoroꢀ1H,1'Hꢀ[2,2']biindolylꢀ3ꢀ
ylmethyl]ꢀpyrrolidinꢀ3Sꢀyl ester (27). To a solution of 21 (0.64 kg, 0.78 mol) in DCM (3 L) was
added HBr solution (33 wt% in acetic acid, 1.1 L, 6.3 mol) at ambient temperature. After 48 h,
the reaction mixture was transferred into a mixture of water (15 L) and MTBE (9 L) at 0ꢀ5 °C.
The batch was stirred for 30 min and the organic phase was removed. The aqueous phase was
washed with MTBE (6.4 L) and charged with 2ꢀMeTHF (13 L) and 2 N NaOH (11 L) to adjust
the pH to 7.8ꢀ8.2 while maintaining the internal temperature below 5 °C. After 15 min, the
organic phase was separated. The aqueous phase was extracted with 2ꢀMeTHF (9.6 L). The
combined organic phase was washed with 5 wt% brine and concentrated. After solvent
exchange with MeOH (6.5 L), the solid was collected by filtration and washed with MeOH (2 ×
0.7 L). The wet cake was dried under vacuum at 40 °C for 16 h to give 27 as an offꢀwhiteꢀ
1
colored solid (0.72 kg, 93% yield, >98 area% by HPLC). H NMR (300 MHz, CDCl3): δ 13.06
(br s, 2H) 7.65 (dd, J = 5.4, 8.7 Hz, 2H), 7.03 (dd, J = 1.8, 9.6 Hz, 2H), 6.87 (m, 2H), 5.37 (m,
2H), 3.74 (m, 2H), 3.18ꢀ3.35 (m, 4H), 2.92ꢀ3.6 (m, 4H), 2.59 (m, 2H), 2.00ꢀ2.20 (m, 2H), 2.05
(s, 6H), 1.80ꢀ1.92 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3): δ 171.0, 159.9 (d, JCF = 235.8 Hz),
135.7(d, JCF = 10.0 Hz), 130.3, 130.2, 125.1, 118.7 (d, JCF = 10.0 Hz), 109.3, 107.5 (d, JCF = 24.3
Hz), 97.4 (d, JCF = 25.1 Hz), 74.9, 57.4, 51.4, 38.4, 32.6, 21.4 ppm. HRMS (ESI), m/z 551.2471
[(M+H), calcd for C30H33F2N4O4: 551.2470].
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Acetic
acid
5Rꢀ{3'ꢀ[4Sꢀacetoxyꢀ1ꢀ(2Sꢀtertꢀbutoxycarbonylaminoꢀbutyryl)ꢀpyrrolidinꢀ2Rꢀ
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ylmethyl]ꢀ6,6'ꢀdifluoroꢀ1H,1'Hꢀ[2,2']biindolylꢀ3ꢀylmethyl}ꢀ1ꢀ(2Sꢀtertꢀbutoxycarbonylaminoꢀ
butyryl)ꢀpyrrolidinꢀ3Sꢀyl ester (29). To a solution containing BocꢀAbuꢀOH (0.76 kg, 3.8 mol)
and HATU (1.6 kg, 4.1 mol) in anhydrous NMP (6.9 L) at 0ꢀ5 ºC was added NMM (0.62 L, 5.6
mol) followed by a solution of 27 (0.86 kg, 1.6 mol) in NMP (5.2 L). The reaction mixture was
warmed to ambient temperature over 2 h. The reaction mixture was diluted with EtOAc (17 L)
and MTBE (9 L) and washed with 1 N HCl (6.9 L). The layers were separated and the aqueous
phase was extracted with 2:1 EtOAc/MTBE (7.8 L). The combined organic phase was washed
successively with saturated NaHCO3 and brine. The 29ꢀcontaining organic phase was
concentrated followed by solvent exchange to DCM (10 L) and used directly in the next reaction.
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An analytical sample was prepared: H NMR (300 MHz, CDCl3), mixture of rotamers: δ 11.38
(br s, 2H), 7.44 (dd, J = 5.1, 15.8 Hz, 2H), 7.37 (d, J = 9.30 Hz, 2H), 6.80 (dd, J = 8.7, 8.7, 2H),
6.88 (br s, 2H), 5.45 (m, 2H), 4.36 (m, 4H), 4.15 (dd, J = 4.2, 12.0 Hz, 2H), 3.80 (d, J = 12.6 Hz,
2H), 3.15ꢀ3.40 (m, 4H), 2.32 (m, 2H), 2.27 (s, 6H), 1.90 (m, 2H), 1.74 (m, 4H), 1.53 (s, 18H),
1.02 (t, J = 7.2 Hz, 6H) ppm; 13C NMR (75 MHz, CDCl3) , mixture of rotamers: δ 172.5, 170.4,
160.0 (d, JCF = 235.8 Hz) , 155.9, 137.1 (d, JCF = 12.9 Hz), 128.6, 125.6, 118.7 (d, JCF = 9.8 Hz),
108.4 (d, JCF = 24.9 Hz), 108.3, 98.3 (d, JCF = 25.9 Hz), 80.6, 74.6, 60.3, 53.8, 53.5, 34.0, 28.7,
28.4, 26.2, 21.6, 10.4 ppm. HRMS (ESI), m/z 921.4598 [(M+H), calcd for C48H63F2N6O10:
921.4574].
Acetic acid 5Rꢀ{3'ꢀ[4Sꢀacetoxyꢀ1ꢀ(2Sꢀaminoꢀbutyryl)ꢀpyrrolidinꢀ2Rꢀylmethyl]ꢀ6,6'ꢀdifluoroꢀ
1H,1'Hꢀ[2,2']biindolylꢀ3ꢀylmethyl}ꢀ1ꢀ(2Sꢀaminoꢀbutyryl)ꢀpyrrolidinꢀ3Sꢀyl ester (33). A solution
containing 29 in DCM (10 L) was added TFA (2.3 L) at the rate that maintained the batch
temperature below 25 °C. The resulting mixture was stirred at ambient temperature for 40 h, then
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slowly transferred to a mixture of 2ꢀMeTHF (52 L) and saturated NaHCO3 (28 L). After 15 min,
the organic phase was separated. The aqueous phase was back extracted with 2ꢀMeTHF (17 L).
The combined organic phase was washed successively with saturated NaHCO3 and brine and
concentrated. The residue was diluted with MTBE (6.9 L) followed by a solvent swap to MTBE
(6.9 L, 2ꢀMeTHF ≤10%) to precipitate 33 which was collected by filtration. The wet cake was
washed with MTBE (1.7 L) and dried under vacuum at 40 °C for 91 h to give crude 33 as pale
yellowꢀcolored solid which was purified by recrystallization in MeOH/2ꢀMeTHF/MTBE to
provide 33 as a whiteꢀcolored solid (0.97 kg, 86% overall yield, >99 area% by HPLC). 1H
NMR (300 MHz, CDCl3 + MeOHꢀd4), mixture of rotamers: δ 7.57 (dd, J = 5.1, 9.9 Hz, 2H), 7.53
(dd, J = 2.4, 7.8 Hz, 2H), 6.89 (ddd, J = 2.4, 9.3, 13.2 Hz, 2H), 5.48 (dd, J = 4.5, 4.5 Hz, 2H),
4.52 (dd, J = 9.3, 9.3 Hz, 2H), 4.06 (dd, J = 5.1, 12.3 Hz, 2H), 3.78 (d, J = 12.3 Hz, 2H), 3.54ꢀ
3.70 (m, 4H), 3.30ꢀ3.40 (m, 2H), 3.14 (s, 6H), 2.34 (m, 2H), 2.33 (s, 6H), 2.10 (m, 2H), 1.86 (m,
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2H), 1.78 (m, 2H), 1.09 (t, J = 7.2 Hz, 6H) ppm; C NMR (75 MHz, CDCl3 + MeOHꢀd4):
δ 173.5, 170.9, 160.2 (d, JCF = 235.8 Hz), 137.2 (d, JCF = 12.8 Hz), 128.2, 128.1, 125.6, 118.7 (d,
JCF = 10.0 Hz), 108.6 (d, JCF = 24.9 Hz), 108.0, 98.4 (d, JCF = 25.7 Hz), 74.6, 60.1, 53.5, 33.5,
28.0, 21.4, 9.7 ppm. HRMS (ESI), m/z 721.3539 [(M+H), calcd for C38H47F2N6O6: 721.3525].
Acetic
acid
5Rꢀ(3'ꢀ{4Sꢀacetoxyꢀ1ꢀ[2Sꢀ(2Sꢀmethylꢀ(tertꢀbutoxycarbonyl)ꢀaminoꢀ
propionylamino)ꢀbutyryl]ꢀpyrrolidinꢀ2Rꢀylmethyl}ꢀ6,6'ꢀdifluoroꢀ1H,1'Hꢀ[2,2']biindolylꢀ3ꢀ
ylmethyl)ꢀ1ꢀ[2Sꢀ(2Sꢀmethylꢀ(tertꢀbutoxycarbonyl)ꢀaminoꢀpropionylamino)ꢀbutyryl]ꢀpyrrolidinꢀ
3Sꢀyl ester (34). To a solution containing BocꢀN(Me)AlaꢀOH (0.65 kg, 3.2 mol) and HATU (1.4
kg, 3.7 mol) in anhydrous NMP (7.6 L) at 0ꢀ5 ºC was added NMM (0.53 L, 4.8 mol) followed by
addition of 33 (0.96 kg, 1.3 mol) in NMP (8.7 L). The resulting mixture was allowed to warm to
ambient temperature. After 3 h, the reaction mixture was diluted with MTBE (29 L) and washed
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successively with water (19 L), 1 N HCl (7.6 L), saturated NaHCO3 (2 × 7.6 L), and brine (5.7
L). The organic phase was separated and concentrated to dryness. The residue was dissolved in
MTBE (7.4 L) at 50 ºC followed by addition of nꢀheptane (5.0 L). After cooling to 0ꢀ5 ºC, 34
was collected by filtration. The wet cake was washed with a 3:2 MTBE/nꢀheptane (2 × 1.5 L)
and dried under vacuum at 30 ºC for 28 h to provide 34 as a whiteꢀcolored solid (1.3 kg, 87%
yield, >99 area% by HPLC). 1H NMR (300 MHz, CDCl3): δ 11.57 (br s, 2H), 7.40ꢀ7.60 (m,
4H), 6.89 (ddd, J = 2.4, 4.5, 9.0 Hz, 2H), 5.50 (dd, J = 5.1, 5.1 Hz, 2H), 4.76 (m, 2H), 4.67 (q, J
= 6.9 Hz, 2H), 4.50 (t, J = 9.6 Hz, 2H), 4.20 (dd, J = 3.9, 12.3 Hz, 2H) 3.85 (d, J = 12.3 Hz, 2H),
3.57 (br d, J = 13.5 Hz, 2H), 3.34 (dd, J = 12.0, 13.8 Hz, 2H), 2.89 (s, 6H), 2.34 (s, 6H), 2.30
(brs, 2H), 2.02ꢀ2.18 (m, 4H), 1.95 (td, J = 6.0, 13.8 Hz, 2H), 1.79 (td, J = 7.2, 14.1 Hz, 2H), 1.52
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55
56
57
58
59
60
13
(s, 18H), 1.39 (d, J = 7.2 Hz, 6H), 1.03 (t, J = 7.2 Hz, 6H) ppm; C NMR (75 MHz, CDCl3):
δ 174.6, 172.2, 172.0, 170.5, 160.3 (d, JCF = 236.4 Hz) , 137.4 (d, JCF = 12.9 Hz), 128.5, 128.4,
125.8, 118.6 (d, JCF = 10.0 Hz), 108.6 (d, JCF = 24.6 Hz), 108.1, 98.6 (d, JCF = 25.7 Hz), 81.1,
74.6, 60.1, 54.0, 52.0, 33.7, 30.5, 28.6, 28.1, 25.9, 21.6, 20.9, 14.0, 9.9 ppm. HRMS (ESI), m/z
1091.5779 [(M+H), calcd for C56H77F2N8O10: 1091.5629].
Acetic acid 5Rꢀ(3'ꢀ{4Sꢀacetoxyꢀ1ꢀ[2Sꢀ(2Sꢀmethylaminoꢀpropionylamino)ꢀbutyryl]ꢀpyrrolidinꢀ
2Rꢀylmethyl}ꢀ6,6'ꢀdifluoroꢀ1H,1'Hꢀ[2,2']biindolylꢀ3ꢀylmethyl)ꢀ1ꢀ[2Sꢀ(2Sꢀmethylaminoꢀ
propionylamino)ꢀbutyryl]ꢀpyrrolidinꢀ3Sꢀyl ester (36). To a solution containing 34 (1.25 kg, 0.70
mol) in DCM (11 L) was added TFA (1.3 L) at a rate maintaining the internal temperature below
25 °C. The resulting mixture was stirred at ambient temperature for 16 h and quenched by
transferring to a mixture of saturated NaHCO3 (38 L) and 2ꢀMeTHF (38 L). The organic phase
was separated and the aqueous phase was extracted with 2ꢀMeTHF (13 L). The combined
organic phase was washed successively with saturated NaHCO3 (6 L) and brine (6 L) and
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concentrated. The residue was dissolved in MeOH (7 L) and the resulting 36ꢀcontaining solution
was used directly in the next reaction. An analytical sample was prepared: 1H NMR (300 MHz,
CDCl3): δ 11.76 (s, 2H), 8.19 (d, J = 8.7 Hz, 2H), 7.52 (dd, J = 2.1, 10.0 Hz, 2H), 7.52 (dd, J =
5.4, 8.9 Hz, 2H), 6.88 (ddd, J = 2.1, 9.2, 9.2 Hz, 2H), 5.48 (dd, J = 4.5, 4.5 Hz, 2H), 4.68 (m,
2H), 4.47 (m, 2H), 4.20 (dd, J = 5.4, 12.5 Hz, 2H), 3.86 (d, J = 12.3 Hz, 2H), 3.61 (dd, J = 1.8,
12.6 Hz, 2H), 3.30 (dd, J = 12.0, 14.1 Hz, 2H), 3.12 (q, J = 6.6 Hz, 2H), 2.56 (s, 6H), 2.34 (s,
6H), 3,27 (br d, J = 15.3 Hz, 2H), 2.07 (m, 2H), 1.97 (m, 2H), 1.84 (m, 2H), 1.71 (br s, 2H), 1.38
10
11
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14
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13
(d, J = 6.9 Hz, 6H), 1.08 (t, J = 7.5 Hz, 6H) ppm; C NMR (75 MHz, CDCl3): δ 175.2, 172.7,
170.5, 160.3 (d, JCF = 235.8 Hz), 137.4 (d, JCF = 12.9 Hz), 128.5 (d, JCF = 3.5 Hz), 125.9, 118.5
(d, JCF = 10.0 Hz), 108.5 (d, JCF = 24.9 Hz), 107.8, 98.6 3 (d, JCF = 25.7 Hz), 74.6, 60.9, 60.0,
53.9, 51.3, 35.8, 33.8, 27.9, 26.1, 21.6, 20.2, 10.0 ppm. HRMS (ESI), m/z 913.4435 [(M+Na),
calcd for C46H60F2N8O8Na: 913.4424].
Nꢀ{1Sꢀ[2Rꢀ(6,6'ꢀDifluoroꢀ3'ꢀ{4Sꢀhydroxyꢀ1ꢀ[2Sꢀ(2Sꢀmethylaminoꢀpropionylamino)ꢀbutyryl]ꢀ
pyrrolidinꢀ2Rꢀylmethyl}ꢀ1H,1'Hꢀ[2,2']biindolylꢀ3ꢀylmethyl)ꢀ4Sꢀhydroxyꢀpyrrolidineꢀ1ꢀ
carbonyl]ꢀpropyl}ꢀ2Sꢀmethylaminoꢀpropionamide (1). To a solution containing 36 in MeOH (13
L) from previous step was added 1 M NaOH (3.5 L) at 0 ºC. The resulting mixture was stirred
for 4 h at ambient temperature. MeOH was removed in vacuo at 35 ºC and the residue was
diluted with water (25 L) and extracted with 2ꢀMeTHF (25 L). The aqueous phase was separated
and extracted with 2ꢀMeTHF (13 L). The combined organic extracts were washed with water (13
L) and concentrated to dryness. The residue was suspended in MTBE (7 L) at 35 °C for 1 h and
the solid was collected by filtration and washed with MTBE (2.6 L). The wet cake was dried
under vacuum at 45 ºC for 16 h to provide 1 as an offꢀwhiteꢀcolored solid (0.96 kg, 99.6 area%
by HPLC). The crude product (0.96 kg) was dissolved in a mixture of EtOAc (14 L), IPA (5 L)
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and water (0.6 L). The volume was reduced by 50% under reduced pressure at 35ꢀ40 ºC. To the
resulting mixture was added EtOAc (29 L) and the solution was concentrated at 35ꢀ40 ºC under
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reduced pressure until the ratio of EtOAc/IPA ≥15 by H NMR analysis and water content
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≤0.2% by KF analysis. The solution was cooled to ambient temperature. After 16 h, the solid was
collected by vacuum filtration, washed with EtOAc (1 L) and dried under high vacuum at 50±5
°C for 24 h to afford 1 as an offꢀwhiteꢀcolored solid (0.87 kg, 94% overall yield, >99 area% by
1
HPLC). [α]25 = +93.1° (c = 1.0, MeOH). H NMR (500 MHz, DMSOꢀd6), major rotamer:
D
δ 11.92 (s, 2H), 8.30 (d, J = 7.8 Hz, 2H), 7.84 (dd, J = 5.5, 8.7 Hz, 2H), 7.47 (dd, J = 2.3, 9.8 Hz,
2H), 6.94 (dt, J = 2.0, 9.2 Hz, 2H), 5.66 (s, 2H), 4.55 (m, 2H), 4.49 (br s, 2H), 4.26 (br s, 2H),
3.91 (dd, J = 4.4, 11.1 Hz, 2H), 3.72 (d, J = 10.9 Hz, 2H), 3.51 (m, 5H), 3.08 (q, J = 6.9 Hz, 2H),
2.34 (s, 6H), 1.88 (m, 6H), 1.76 (m, 2H), 1.16 (d, J = 6.9 Hz, 6H), 1.00 (t, J = 7.4 Hz, 6H) ppm;
13C NMR (125 MHz, DMSOꢀd6): δ 174.1, 172.0, 159.3 (d, JCF = 235.2 Hz), 136.5 (d, JCF = 13.2
Hz), 127.6 (d, JCF = 3.5 Hz), 125.7, 120.0 (d, JCF = 10.0 Hz), 108.7, 107.8 (d, JCF = 24.5 Hz), 97.5
(JCF = 25.8 Hz), 70.1, 59.2, 59.2, 55.9, 51.2, 35.7, 34.3, 27.6, 24.8, 18.8, 9.6 ppm. HRMS (ESI),
m/z 807.4394 [(M+H), calcd for C42H57F2N8O6: 807.4369].
SUPPORTING INFORMATION
The supporting information contains data refinement for the crystallographic representation of 1,
details on the choice of coꢀsolvent for the addition of 6Fꢀindoylmagnesium bromide to 11 (Table
S4), details on catalyst screening for the hydrogenation of 21 (Table S5), and a description of the
heterodimer synthetic strategy that allowed independent synthesis of 30. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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