Synthesis of four isotopically labeled forms of a proteasome inhibitor, bortezomib

Article abstract of DOI:10.1002/jlcr.1173

[D₂](1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]-amino]butyl] boronic acid ([D₂]bortezomib), a proteasome inhibitor, was synthesized in 11 steps from isobutyryl chloride. Key steps in the synthesis inclu

Full text of DOI:10.1002/jlcr.1173

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 402–406.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1173
Short Research Article
Synthesis of four isotopically labeled forms of a proteasome
inhibitor, bortezomiby
YUEXIAN LI1,*, MIHAELA PLESESCU1, PATRICK SHEEHAN2, J. SCOTT DANIELS3 and SHIMOGA R. PRAKASH1
1 Department of Drug Metabolism and Pharmacokinetics, Millennium Pharmaceuticals, Inc., 35 Landsdowne Street, Cambridge, MA 02139, USA
2 Suffolk University, 8 Ashburton Place, Boston, MA 02108, USA
3 Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., St. Louis, MO 63017, USA
Received 29 August 2006; Revised 6 November 2006; Accepted 21 November 2006
Abstract: [D2](1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]-amino]butyl] boronic acid
([D2]bortezomib), a proteasome inhibitor, was synthesized in 11 steps from isobutyryl chloride. Key steps in the
synthesis included formation of the isobutyryl boronic acid via Grignard reaction and preparation of the chiral
chloride using Matteson reaction. [13C9]bortezomib, [D5]bortezomib, and [D1]bortezomib were similarly synthesized
from appropriate labeled precursors. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: [D2]isobutyl alcohol; [D2]isobutyl bromide; [D2]isobutyl boronic acid; [D1](aS,3aS,4S,6S,7aR)-hexahydro-3a,5,
5-trimethyl-a-(2-methylpropyl)-4,6-methano-1,3,2-benzodioxaborole-2-methanamine; [D2](aS,3aS,4S,6S,7aR)-hexahydro-3a,5,5-
trimethyl-a-(2-methylpropyl)-4,6-methano-1,3,2-benzodioxaborole-2-methanamine; [13C9]bortezomib; [D1]bortezomib; [D2]bortezo-
mib; [D5]bortezomib
Introduction
biotransformation and pharmacokinetic studies.
[D1]bortezomib 5C was synthesized to aid in the
elucidation of the mechanisms mediating the P450-
catalyzed deboronation of bortezomib. [D2]Bortezomib
5D was required to assist in determination of the
structures of two metabolites. [13C9]Bortezomib 5A and
[D5]bortezomib 5B were prepared as internal standards
for the liquid chromatographic/tandem mass spectro-
metric (LC/MS/MS) bioanalysis of nonclinical and
clinical studies.
The 26S proteasome is a multicatalytic proteolytic
complex located in both the nuclei and cytoplasm of
eukaryotic cells.1 The proteasome hydrolyzes proteins
(e.g. misfolded proteins) marked for degradation
through the ubiquitin pathway and plays an essential
role in cellular homeostasis and cell cycle regulation.2
Importantly, various hematological cancers are depen-
dent on the amplified proteasome activity for cell
survival. While the reasons are not completely under-
stood, healthy cells do not exhibit this same depen-
dency, and therefore, inhibition of the proteasome may
represent a therapeutic advantage in targeted cancer
therapy.3 Bortezomib 5 (VELCADE1, formerly known
as PS-341, Scheme 1) is a first-in-class proteasome
inhibitor that has been approved in Europe and the
United States for the treatment of patients with
relapsed multiple myeloma.4 Four isotopically labeled
forms of bortezomib were prepared to support multiple
Results and discussion
Synthesis of [13C9]bortezomib 5A and [D5]bortezomib
5B
Preparation of [13C9]bortezomib 5A: Commercially avail-
able BOC-[13C9]L-phenylalanine 2A was chosen as the
labeled starting material (Scheme 1). The boronate
ester 1 was prepared by the procedure of Kettner et al.5
Coupling of 1 with 2A followed by deprotection of the
resulting 3A led to the amine which was further
coupled with 2-pyrazine carboxylic acid to afford 4A.
Compared to HATU and EDCI, TBTU was found to be
the best coupling agent for these two coupling steps.6,7
*Correspondence to: Yuexian Li, Department of Drug Metabolism and
Pharmacokinetics, Millennium Pharmaceuticals, Inc., 35 Landsdowne
Street, Cambridge, MA 02139, USA. E-mail: yli@mpi.com
y
Proceedings of the Ninth International Symposium on the Synthesis
[
13C9]bortezomib 5A was prepared by the acidic
catalytic transesterification of 4A. The overall yield of
and Applications of Isotopically Labelled Compounds, Edinburgh,
16–20 July 2006.
Copyright # 2007 John Wiley & Sons, Ltd.

SYNTHESIS OF FOUR ISOTOPICALLY LABELED FORMS OF A PROTEASOME INHIBITOR 403
5A was 49%. [D5]bortezomib 5B was prepared in a
chemotherapeutic agent.9 A near equal mixture of
diastereomeric carbinolamide metabolites (M1 and
M2) was observed following metabolism in human
hepatic microsomes (Scheme 2). Metabolites M23 and
M24 were observed at minor levels and were tentatively
assigned as bearing an unsaturation of the isobutyl
side chain.9
similar manner using commercially available BOC-
[D5]L-phenylalanine 2B. The overall yield of 5B was
14%. The lower yield of 5B compared to 5A was due to
the inefficient crystallization of the final compound. 5B
was found to be equally effective compared to 5A as an
internal standard for bioanalytical studies.
In order to assist in the structural elucidation of M23
and M24, [D2]bortezomib 5D was prepared. Isobutyryl
chloride 6 was reduced by lithium aluminum deuteride
to provide [D2]isobutyl alcohol (Scheme 3).10 Bromina-
tion of this alcohol with PBr3 yielded [D2]isobutyl
bromide 7.11 The low yield of this bromination step
was due to the decomposition of 7 during its distilla-
tion. The reaction of Grignard 7 with trimethylborate,
Synthesis of [D2]bortezomib 5D and [D1]bortezomib 5C
Inhibition of the 26S proteasome involves a dative bond
between the N-terminal threonine residue of the
proteasome and the boron atom of bortezomib.3,8
Hence, deboronation represents a deactivation path-
way and is the principal route of metabolism of this
Z
Z
Z
Z
Z
Z
Z
Z
*
TFA-
+H3N
*
TBTU/DIPEA
DMF/00C
O
O
*
O
Z
NH
O
O
B
*
O
Z
OH
B
+
O
NH
*
*
87-91%
*
NH
2A:*=13C, Z=H
2B:*=12C, Z=D
O
*
O
3A: *=13C, Z=H
3B: *=12C, Z=D
O
1
Z
Z
*
OH
OH
Z
Z
Z
Z
Z
Z
1. HCl/EtOAc
2. TBTU/DIPEA
DMF/00C
B
*
*
*
O
O
Z
NH
Z
O
O
OH
OH
O
HCl/MeOH
Hexanes/rt
NH
N
N
N
N
B
B
N
*
N
*
N
OH
*
*
H
H
O
O
N
36-74%
5A: *=13C, Z=H
5B: *=12C, Z=D
5: *=12C, Z=H
4A:*=13C,Z=H
4B:*=12C,Z=D
44-74%
Scheme 1
O
OH
OH
Human
Microsomes
O
O
NH
B
N
N
NH
NH
OH
OH
N
N
N
H
N
N
H
H
O
O
O
N
N
Bortezomib, 5
+
M2
M1
O
NH
N
N
N
H
-2H
O
M23, M24
Scheme 2
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 402–406
DOI: 10.1002.jlcr

404 Y. LI ET AL.
1. Mg/ether
2. B(OMe)3
3. H2SO4
(i-Pr)2NLi
CY2Cl2
ZnCl2/THF
OH
OH
HO
HO
1. LiAlD4
ether/rt
2. PBr3
-100C
O
O
Cl
B
Br
B
X
MTBE/00C
60-99%
X
X
O
6
X
X
O
61%
X
97-99%
9C: X=H
9D: X=D
8C: X=H
7: X=D
18%
8D: X=D
O
O
O
1. (Me3Si)2NLi
TFA-
+H3N
O
B
THF/-300C
2. CF3COOD
ether/00C
OH
B
Cl
O
O
O
N
O
H
O
Y
NH
X
B
Y
X
X
X
N
H
O
TBTU/DIPEA
DMF/00C
91-99%
X
Y
O
3C: X=H, Y=D
10C: X=H, Y=D
10D: X=D, Y=H
1C: X=H, Y=D
1D: X=D, Y=H
62-80%
X
3D: X=D, Y=H
1. HCl/EtOAc
2. TBTU/DIPEA
DMF/00C
O
OH
B
OH
O
O
B
O
Y
O
OH
OH
NH
X
NH
B
N
N
N
H
N
N
OH
HCl/MeOH
Hexanes/rt
H
Y
O
O
X
N
N
N
X
X
4C: X=H, Y=D
4D: X=D, Y=H
5C: X=H, Y=D
5D: X=D, Y=H
39-45%
62-80%
Scheme 3
Cl
Cl
O
Zn
O
(i-Pr)2NLi
CH2Cl2
Cl2HC
-
B
B
O
Cl
O
O
ZnCl2
O
B
Cl
H
-
B
O
Cl
C
O
9C
11
12
H
10
Scheme 4
followed by subsequent hydrolysis, resulted in the
formation of the desired [D2]isobutyl boronic acid,
8.12 Coupling of 8 with (1S,2S,3R,5S)-(+)-pinanediol
afforded the boronate ester 9. Capitalizing on the
exceptional enantioselectivity of the Matteson rearran-
gement, the reaction of the boronate ester (9) with
dichloromethane and ZnCl2 provided the key chiral
chloride 10D in an excellent yield (99%).13,14 Nucleo-
philic substitution of 10D with lithium hexamethyldi-
silazide followed by the deprotection (TFA) of two
trimethylsilyl groups afforded the chiral amine 1D.
The subsequent four steps followed the same methods
described in Scheme 1 to get the final product 5D,
[D2]bortezomib. The overall yield of the 11-step synth-
esis of 5D was 1%.
The enantioselectivity of the Matteson reaction may
be explained by the formation of two intermediates
(Scheme 4).15 The first intermediate 11 is formed by
coordinating the Lewis acid (the boronate ester 9C) with
the carbanion produced from CH2Cl2. The second
intermediate 12 is formed by subsequent coordination
of a Lewis acid catalyst (ZnCl2) with an oxygen and a
chlorine atom of 11. The enantioselectivity one ob-
serves from the Matteson rearrangement may be best
explained via the proposed transition state chemistry
depicted in Scheme 4. The spacial arrangement
of coordinate complex 12 is such that the critical
carbon-bond migration is favored, thus resulting in the
desired precursor, 10.
[D1]bortezomib 5C was instrumental in the mechan-
istic investigation into the unusual P450-catalyzed
deboronation of bortezomib. Compound 5C was
synthesized in a manner analogous to the later stage
preparation of [D2]bortezomib 5D. The overall yield of
the eight-step synthesis of 5C was 9%.
Mechanism of P450-catalyzed deboronation of
bortezomib
There are two possible mechanisms to explain the
P450-catalyzed deboronation of bortezomib.9 The first
mechanism is the peroxidation by iron-peroxo species
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 402–406
DOI: 10.1002.jlcr

SYNTHESIS OF FOUR ISOTOPICALLY LABELED FORMS OF A PROTEASOME INHIBITOR 405
N
N
Fe
N
N
O
O
O
O
OH
OH
O
NH
OH
OH
NH
N
N
-
B
NH
peroxo-iron
P450
N
N
B
N
N
N
H
N
H
N
H
OH
D
D
O
D
O
O
[D1]-M1
bortezomib
Scheme 5
O
NH
OH
N
N
N
H
reactive
oxygen
D
O
O
N
OH
OH
O
species
(ROS)
[D1]-M1
O
N
NH
NH
B
OOH
N
N
O2
NH
reduction
N
H
.
D
N
H
N
D
D
O
O
P450
H
O
O
N
Bortezomib, 5
NH
N
OH
N
N
N
H
D
O
[D1]-M2
Scheme 6
produced in the P450 catalytic cycle (Scheme 5), where
the iron-peroxo mediated reaction would result in the
stereocontrolled oxidation of 5 (i.e. retention of stereo-
chemistry at the migrating carbon center producing a
single diastereomer). Consequently, two metabolites
(M1 and M2) were produced as a result of human
metabolism of 5. These data would indicate an alter-
native mechanism contributing to the deboronation of
this chemotherapeutic agent. Importantly, incubation
of isolated carbinolamide M1 with human liver micro-
somes did not afford a mixture of M1 and M2;9 this
finding eliminated the possible role of reversible
dehydration as a means of producing the metabolite
mixture.
human liver metabolism of 5C resulted in the same
metabolite profile, with no apparent alteration of the
ratios of M1 or M2. Furthermore, no loss of deuterium
label was observed during the metabolism of 5C, thus
providing additional evidence for a homolytic-autoxi-
dation mechanism mediating the deboronation of 5
(Daniels JS et al. unpublished results).16,17
Conclusion
In summary, a practical method for the preparation of
the D1 chiral amine 1C and D2 chiral amine 1D was
developed. Grignard reaction of D2 isobutyl bromide 7
with trimethyl borate followed by hydrolysis provided a
convenient method of preparing D2 isobutyl boronic
acid 8D in a satisfactory yield. The Matteson reaction of
9D (or 9C) with CH2Cl2 (or CD2Cl2) and ZnCl2 provided
a practical method of preparing D2 chiral chloride 10D
(or D1 chiral chloride 10C) in an excellent yield.
Chemical purities of labeled compounds were assessed
by HPLC, LC/MS/MS, and NMR.
Given the numerous historical references of boronate
autoxidation, another probable mechanism considered
was deboronation via the varied reactive oxygen species
(ROS) produced as byproducts of P450 catalysis
(Scheme 6).9 Formation of an epimerizing carbon-
centered radical is implicated in the lack of stereo-
chemical control historically observed in the autoxida-
tion of boronates, i.e. the stereochemical approach of
molecular oxygen with the said radical is indiscrimi-
nate. Importantly, the autoxidation mechanism is
proposed to be initiated via the homolytic fragmenta-
tion of the carbon-boron bond. Therefore, substitution
of deuterium at the alpha boronate carbon should have
no effect on the overall deboronation reaction. Indeed,
Acknowledgements
Thanks are due to the Process Chemistry Research and
Development of Millennium Pharmaceuticals for the
provision of unlabeled intermediates.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 402–406
DOI: 10.1002.jlcr

406 Y. LI ET AL.
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J Label Compd Radiopharm 2007; 50: 402–406
DOI: 10.1002.jlcr