Discovery of a potent, selective, and orally active proteasome inhibitor for the treatment of cancer

Article abstract of DOI:10.1021/jm7010589

The ubiquitin-proteasome pathway plays a central role in regulation of the production and destruction of cellular proteins. These pathways mediate proliferation and cell survival, particularly in malignant cells. The successful development of the 20S human proteasome inhibitor bortezomib for the treatment of relapsed and refractory multiple myeloma has established this targeted intervention as an effective therapeutic strategy. Herein, the potent, selective, and orally bioavailable threonine-derived 20S human proteasome inhibitor that has been advanced to preclinical development, [(1R)-1-[[2S,3R)-3-hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]-1-oxobutyl] amino]-3-methylbutyl]boronic acid 20 (CEP-18770), is disclosed.

Full text of DOI:10.1021/jm7010589

1068
J. Med. Chem. 2008, 51, 1068–1072
Discovery of a Potent, Selective, and Orally Active Proteasome Inhibitor for the Treatment of
Cancer
Bruce D. Dorsey,*,† Mohamed Iqbal,† Sankar Chatterjee,† Ernesto Menta,‡ Raffaella Bernardini,‡ Alberto Bernareggi,‡
Paolo G. Cassarà,‡ Germano D’Arasmo,‡ Edmondo Ferretti,‡ Sergio De Munari,‡ Ambrogio Oliva,‡ Gabriella Pezzoni,‡
Cecilia Allievi,‡ Ivan Strepponi,‡ Bruce Ruggeri,† Mark A. Ator,† Michael Williams,† and John P. Mallamo†
Cephalon, Inc., 145 Brandywine Parkway, West Chester, PennsylVania 19380, and Cell Therapeutics Europe S.r.l., Via L. Ariosto,
23, I-20091 Bresso, Italy
ReceiVed August 27, 2007
The ubiquitin-proteasome pathway plays a central role in regulation of the production and destruction of
cellular proteins. These pathways mediate proliferation and cell survival, particularly in malignant cells.
The successful development of the 20S human proteasome inhibitor bortezomib for the treatment of relapsed
and refractory multiple myeloma has established this targeted intervention as an effective therapeutic strategy.
Herein, the potent, selective, and orally bioavailable threonine-derived 20S human proteasome inhibitor
that has been advanced to preclinical development, [(1R)-1-[[(2S,3R)-3-hydroxy-2-[(6-phenylpyridine-2-
carbonyl)amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid 20 (CEP-18770), is disclosed.
Introduction
The ubiquitin-proteasome pathway (UPP) is responsible for
most intracellular degradation of proteins in eukaryotes. Within
the cytosol and nucleus, ubiquitin is activated by an ubiquitin-
activating enzyme (E1) and transferred to an ubiquitin-conjugat-
ing enzyme (E2). Specific binding of E2 enzyme with sub-
strate–protein to an ubiquitin-protein ligase (E3) permits the
transfer of a polyubiquitin chain. Once tagged, these polyubiq-
uitin proteins are then degraded by 26S proteasome into
ubiquitin and short peptides that are further processed into
recyclable amino acids.1,2 The proteolytic component of the UPP
consists of a multicatalytic protein core particle (CP) known as
the 20S proteasome capped by two 19S regulatory particles
(RPs). The RPs are responsible for recognition, unfolding, and
translocation of protein–substrates into the CP cavity where
proteolytic processing occurs. Crystal structures of eukaryotic
20S proteasomes reveal an elongated shaped cylinder for the
CP, which contains four stacked rings. Each ring is composed
of seven different R and ꢀ subunits where the outer R rings of
the complex interact with the RPs and the two inner ꢀ rings
contain the proteolytically active sites.3 Three different active
sites are responsible for the postglutamyl (ꢀ1), tryptic (ꢀ2), and
chymotryptic (ꢀ5) proteolytic activities.4 Each utilizes the
nucleophilic γ-hydroxyl group of the N-terminal threonine (Thr)
to initiate amide bond hydrolysis followed by activation of
nucleophilic water by the R-amine of Thr to hydrolyze the
resulting ester.
Among the various enzymatic activities of the proteasome,
the “chymotrypsin-like” activity has emerged as the biological
function of greatest interest and focus of drug discovery efforts.
Increased levels of this enzyme and subsequent protein break-
down have been implicated in many disease states including
muscular dystrophy, emphysema, cancers such as acute myeloid
leukemia, and cachexia accompanying cancer and AIDS.5
Proteasome function also controls levels of proteins critical for
cell cycle control, including p53, p27, and cyclin B and is
Figure 1. Structures of bortezomib, vinyl ester tripeptides, epoxomicin,
reversible 2-keto-1,3,4-oxadiazoles, and the natural product salino-
sporamide A.
responsible for activation of the transcription factor NF-κB
through the degradation of its regulatory subunit IκBR.6 Thus,
development of proteasome inhibitors has emerged as an
attractive target for cancer therapy.7,8 Clinical validation of this
approach has been demonstrated with bortezomib 1 (Figure 1),
a proteasome inhibitor recently approved as a single agent for
the treatment of patients with relapsed and refractory multiple
myeloma with at least one prior line of therapy and for the
treatment of mantle cell lymphoma.9,10 Although this drug has
demonstrated higher overall response rates and prolonged
survival compared to standard treatments, such as dexametha-
sone, some liabilities exist. Bortezomib is administered as an
intravenous bolus, and dose-limiting toxicities, adverse events,
and resistance to single agent therapy have been reported.11
Therefore, there remains a significant need to develop new
proteasome inhibitors with potentially unique properties, includ-
ing activity following oral administration, different proteasome
inhibition profile, and greater therapeutic index.
* To whom correspondence should be addressed. Phone: (610) 738-6472.
Fax: (610) 738-6643. E-mail: bdorsey@cephalon.com.
†
Cephalon, Inc.
Cell Therapeutics Europe S.r.l.
‡
10.1021/jm7010589 CCC: $40.75  2008 American Chemical Society
Published on Web 02/05/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1069
Table 1. Proteasome Inhibitory Activity
compd
R
HEP IC50 (SD), nM
CHY IC50 (range µM), µM
Molt-4 EC50 (SD), nM
A2780 IC50 (SD), nM
15
16
17
18
19
20
21
22
23
24
25
1
pyrazin-2-yl
naphthalen-2-yl
quinolin-2-yl
4-biphenyl
3-biphenyl
6-phenylpyridin-2-yl
4-phenylpyridin-2-yl
3-phenyl-pyridin-2-yl
5-phenylpyrazin-2-yl
6-phenylpyrazin-2-yl
2-phenyl-thiazol-4-yl
bortezomib
42.0 (2.1)
1.6 (0.1)
6.8 (0.4)
0.9 (0.2)
0.8 (0.1)
3.8 (1.0)
2.0 (0.1)
8.8 (1.1)
1.8 (0.1)
2.3 (0.2)
4.6 (0.4)
3.8 (0.4)
ND
1353 (19.2)
27 (6)
32 (8.5)
ND
0.5 (0.3–0.8)
1.30 (1.0–1.8)
0.6 (0.3–0.9)
0.9 (0.8–1.0)
1.5 (0.9–1.8)
0.29 (0.2–0.4)
0.46 (0.3–0.6)
0.27 (0.2–0.4)
0.28 (0.3–0.5)
0.59 (0.5–0.61)
0.5 (0.3–0.9)
5.3 (3.1)
20.9 (7.5)
4.9 (1.2)
4.8 (4.0)
13.7 (2.3)
5.1 (2.7)
ND
8.2 (2.9)
9.8 (2.9)
28.0 (3.8)
1.7 (0.4)
21 (1.4)
16.9 (5.4)
13.5 (2.0)
18.6 (1.0)
13000 (100)
21 (5.0)
27 (6.0)
16 (3.0)
21 (3.0)
Scheme 1. Generalized Synthesis of Dipeptidylboronic Acidsa
Several years ago, starting from a tetrapeptide aldehyde
inhibitor, novel, potent, and selective dipeptide aldehyde inhibi-
tors of the chymotrypsin-like activity of the proteasome were
identified.12 Subsequently, 12 (Table 1) and related analogues
emerged as a useful tools in proteasome research.13 Other
research groups have disclosed reversible and irreversible
peptide-based inhibitors of the 20S proteasome. The vinyl ester
inhibitor 2 (Figure 1) represents a series of irreversible inhibi-
tors14 with submicromolar trypsin-like activity and limited
chymotrypsin-like activity. Conversely, Crews et al.15 reported
irreversible epoxomicin analogues (3) with ꢀ1 and ꢀ5 selectivity
that demonstrate the independent nature of the active site
functions. Potent, selective, and reversible inhibitors of the
chymotrypsin-like activity possessing a unique keto-1,3,4-
oxadiazole warhead have been reported by Rydzewski.16
Represented by 4, this subnanomolar inhibitor is active against
the proteasome contained in PC3 cells, a human prostate cancer
cell line. A recently discovered natural product, salinosporamide
A (5) from the marine microorganism Salinispora tropica, has
been reported that displays nanomolar potency against the 20S
proteasome17 and oral antitumor activity in xenograft model
studies.18
a Reagents and conditions: (a) Et2O, room temp, 24 h; (b) (i) CH2Cl2,
THF, LDA, -78°C; (ii) 1.0 M ZnCl2 in ether, -78°C; (c) LiN(SiMe3)2,
THF, -78 °C to room temp; (d) HCl in dioxane, ether, 0°C; (e) t-Boc-L-
threonine, NMM, TBTU, DMF, 0°C; (f) RCOOH, TBTU, NMM, DMF,
0°C; (g) isobutylboronic acid, 2 N HCl, MeOH, hexane.
1H NMR. Coupling of amine 12 to various acids followed by
acid catalyzed ester exchange with isobutylboronic acid provided
target compounds 13–25 (44–75% yield for two steps).
Biological Results. Proteasome inhibitory activity of the
target compounds was evaluated in an isolated 20S human
erythrocyte proteasome (HEP) fluorimetric kinetic assay of
chymotrypsin-like proteasome activity.12 Activity of the com-
pounds was also investigated in the human leukemia cell line
Molt-4 using the fluorogenic chymotrypsin-like cell permeable
fluorogenic substrate MeOSuc-FLF-AFC, following a published
procedure; thus, this assay also served as a measure of cell
permeability of an inhibitor.13 Determination of cross-reactivity
against human pancreas R-chymotrypsin (CHY) using a fluo-
rogenic specific substrate was also assessed. Table 1 displays
the biological data, expressed as IC50 values for HEP and CHY
assays, and EC50 values for Molt-4. The antiproliferative activity
was determined in A2780 ovarian carcinoma cell line using
standard colorimetric assay to assess cell proliferation and
cellular viability. Additional selectivity data were obtained
against a panel of proteases representing all mechanistic classes
of proteases (see Supporting Information, Table S1).
In our continued search for a potent and druglike proteasome
inhibitor, additional boronate-derived small-molecule inhibitors
were explored. Building on previous advances, potent boronic
esters were identified. Ultimately, this led to identification of
an orally active proteasome inhibitor that is currently being
evaluated for development opportunities.
Results and Discussion
Synthesis. The generalized preparation of dipeptidyl boronic
acids, specifically (2S,3R)-2-amino-3-hydroxybutyric acids rep-
resented by 14, is illustrated in Scheme 1. Following a procedure
of Matteson,19 benzodiozaborole 8 was obtained in 92% yield
over two steps and in high enantiomeric purity from com-
mercially available starting materials. Treatment of benzodiox-
aborole 8 with lithium bis(trimethylsilyl)amide produced pro-
tected amine 9 that underwent facile deprotection in HCl to
generate the desired amino boronate 10 as the hydrochloride
salt in 66% overall yield on a 50 g scale.20 Amino boronate 10
was coupled with t-Boc-L-threonine to provide, after chromato-
graphic purification, amide 11 which was deprotected with
hydrochloric acid in dioxane to produce the versatile intermedi-
ate 12 in 50% yield. Only one diastereomer was detected by
Discussion
As summarized in Table 1, initial attempts to identify a
superior small-molecule proteasome inhibitor involved modi-
fication of the previously reported boronic ester 13.12 Amino

1070 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Brief Articles
Table 2. Single Dose iv and po Pharmacokinetic Profiles of 20 and 1 in
Rats and Mice
acid scanning of the P2 position, including phenylalanine,
glycine, 2,3-diaminoproprionic acid, citrulline, and asparagine,
resulted in the replacement of the nitroarginine moiety in 13
with threonine. Subsequent exploration of the P3 position with
small, focused libraries revealed that replacement of branched
alkyl moieties with naphthyl, quinolyl, or biaryl ligands provided
the required potency and elimination of a chiral center. All
compounds inhibited the chymotrypsin-like activity of 20S
proteasome with IC50 values from 0.8 to 42 nM. Initially, to
benchmark our efforts against bortezomib,21 the pyrazine 15,
in which the P2 ligand threonine replaced phenylalanine, was
prepared. Despite the structural similarity, this resulted in an
11-fold loss in enzyme potency and greater than 50-fold loss
in cellular activity for 15 versus 1. This result focused our efforts
on substituted phenyl rings and heterocyclic biaryl moieties.
The fused bicyclic aryls (i.e., 16 or 17) or the 4- or 3-substituted
biphenyl (18, 19) analogues exhibited excellent enzyme, cellular,
and antiproliferation activities. Of note, the 3-biphenyl 19
inhibited pancreatic R-chymotrypsin with IC50 ) 900 nM,
demonstrating a greater than 1000-fold selectivity for the 20S
proteasome versus bovine R-chymotrypsin activity. This selec-
tivity trend continued with the 6- and 4-phenyl substituted
pyridylcarboxamide analogues 2022 and 21, respectively. Erosion
of this selectivity was observed for the 3-phenyl substituted
pyridine 22. This phenyl substituted regioisomer has HEP IC50
) 8.8 nM and CHY IC50 ) 460 nM with only 52-fold selectivity
for the 20S proteasome. A loss in cellular potency was observed
(Molt-4 EC50 ) 13 000 nM), and further ortho substituted
analogues were not pursued. Modifications of the P3 ligand with
phenyl substituted pyrazines (23, 24) or 2-phenyl-4-thiazole-
carboxamide 25 provided molecules with comparable enzyme
and cellular potency but with somewhat diminished selectivity
versus R-chymotrypsin.
dose
t
AUC0f∞
Vss
CL
F
½
compd
species
S-D rat
(mg/kg) (h) (ng·h/mL) (L/kg) ((L/h)/kg) (%)
20
0.2 iv 71
0.8po 86
1030
2200
2910
2850
927
18.1
10.3
0.19
1.4
54
39
11
CD-1 mice
4
iv 15
10 po 53
CD-1 mice 0.8 iv 98
po 70
1
102
0.86
4
499
phase half-life is due in part to the large volume of distribution,
Vss ) 18.1 L/kg in rats and 10.3 L/kg in mice, and very low
systemic clearance in rats and mice (0.19 and 1.4 (L/h)/kg,
respectively). This experimental observation is consistent with
the lipophilicity, protein binding, and in vitro metabolic stability
in rat and murine liver microsomes (see Supporting Information
Tables S2 and S3). The absolute oral bioavailabilities of 20 in
rats and mice were 54% and 39%, respectively. The bioavail-
ability and systemic clearance data in relation to hepatic blood
flow in rodents suggest that limited first pass metabolism of 20
and bortezomib occurs in the liver. The lower oral bioavailability
of bortezomib is possibly due to reduced absorption in the mouse
gastrointestinal tract. Previous studies have shown that bort-
ezomib is orally bioactive23,24 in models of antigen-induced
arthritis and lung carcinoma xenografts. However, the current
bortezomib therapy in multiple myeloma is administered
intravenously. Another proteasome inhibitor, 5, has recently been
reported to demonstrate comparable activity to bortezomib in a
human subcutaneous plasmacytoma xenograft model after oral
dosing.18 The pharmacokinetic profiles for 20 and bortezomib
in rodents were characterized by prolonged exposure in the
systemic circulation with a long terminal phase half-life, low
systemic clearance, and a large volume of distribution. Despite
the similarity in biochemical proteasome inhibition and subunit
binding kinetics of 20 and bortezomib in normal and tumor cell
lysates, 20 at its iv maximum tolerated dose (MTD) in mice
demonstrated 86% inhibition of proteasome ꢀ5/ꢀ1 subunit
binding in subcutaneous human RPMI 8226 xenograft tissues
in vivo in contrast to that observed with bortezomib (48%
maximum inhibition of ꢀ5/ꢀ1 subunit binding) at its corre-
sponding i.v. MTD in mice.25 Also, 20 and bortezomib showed
comparable potency against chymotrypsin-like proteaseome
activity but demonstrated marginal inhibition of the tryptic and
peptidyl glutamyl activities of the protoeasome.
The selectivity of pyridyl analogue 20 and bortezomib was
assessed in a broad panel of 42 protease assays, representing
all protease mechanistic classes. While most serine proteases
were not affected, modest inhibition of cathepsin G, chymase,
and chymotrypsin was observed for both compounds (Support-
ing Information, Table S1), with IC50 values 150- to 1500-fold
greater than their proteasome chymotrypsin-like inhibitory
activities. Some inhibition of neutrophil elastase 2 was also
detected for 20. No inhibition of cysteine proteases or the
mechanistically distinct metalloproteases and aspartyl proteases
was observed.
To further probe the role proteasome plays in cell viability
and proliferation, the cytotoxic effects of the compounds on
cell growth of a human ovarian CA solid tumor A2780 were
accessed. Importantly, two of the most active compounds in
the Molt-4 cellular assay, 3-biphenyl 19 (EC50 ) 16.9 nM) and
pyridyl 20 (EC50 ) 13.5 nM), were also potent in an antipro-
liferation assay with IC50 of 4.8 and 13.7 nM, respectively. The
4-fold loss of potency from enzyme to cell for 20 is minimal,
demonstrating excellent cell membrane permeation for this
analogue.
To further profile the in vitro characteristics of 20, the in
vitro microsomal metabolic stability, protein binding, and
cytochrome P-450 inhibition were assessed in comparison to
bortezomib. Stability in microsomes was measured, and this
provided an indication of potential phase I oxidative metabolism
(see Supporting Information, Table 2S). An assessment of
cytochrome P-450 enzyme inhibition, indicative of potential
drug-drug interactions, was also conducted using recombinant
human P-450 enzymes and fluorescent P-450 substrates. Inhibi-
tion of the major P-450 isoforms 1A2, 2C9, 2C19, and 2D6 at
concentrations up to 30 µM was not observed; however, 20 and
bortezomib did inhibit 3A4 (IC50 of 3.5 and 17 µM, respec-
tively). Determination of plasma protein binding by ultracen-
trifugation, analyzed by high-throughput liquid chromatography
coupled to a mass spectrometer (see Supporting Information,
Table 3S) revealed that both inhibitors were protein bound across
species. However, the free fraction of 20 was significantly lower
than bortezomib in the presence of human serum albumin. These
data suggest that higher plasma exposure of compound 20 may
These compounds were further differentiated by profiling
several for iv and oral pharmacokinetics in female Spra-
gue–Dawley rats and CD-1 mice. Of the compounds described
in Table 1, 20 demonstrated superior pharmacokinetic param-
eters, and a comparison with bortezomib is illustrated in Table
2.
When dosed in female Sprague–Dawley rats, 20 was slowly
eliminated with estimated terminal half-lifes of 71 and 86 h
after iv and oral administration, respectively, and 15 and 53 h,
respectively, in CD-1 mice (Table 2). This long elimination

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1071
concentrated to provide crude oil. Flash column chromatography
on silica gel using EtOAc/hexane gradient (30%) afforded 257 mg
(44%) of product as a white solid. 1H NMR (DMSO-d6, 400 MHz)
δ 8.98 (d, J ) 2.99 Hz, 1H), 8.76 (d, J ) 8.55 Hz, 1H), 8.2 (m,
3H), 8.11 (t, J ) 7.71 Hz, 1H), 8.02 (d, J ) 7.54 Hz, 1H), 7.54
(m, 3H), 5.26 (d, J ) 4.95 Hz, 1H), 4.49 (dd, J ) 8.52, 4.22, Hz,
1H), 4.13 (m, 2H), 2.6 (m, 1H), 2.19 (m, 1H), 2.02 (m, 1H), 1.83
(t, J ) 5.38 Hz, 1H), 1.75 (s, 1H), 1.68 (m, b, 1H), 1.62 (d, J )
13.9 Hz, 1H), 1.36 (d, J ) 10.05 Hz, 1H), 1.3 (m, b, 3H), 1.22 (d,
J ) 11.65 Hz, 6H), 1.12 (d, J ) 6.26 Hz, 3H), 0.84 (d, J ) 6.57
Hz, 6H), 0.79 (s, 3H).
be required in patients to achieve a comparable therapeutic
benefit to bortezomib.
Conclusion
In this work, (2S,3R)-2-amino-3-hydroxybutyric acid deriva-
tives designed and synthesized as inhibitors of the 20S human
erythrocyte proteasome are disclosed. Prototypes of these
dipeptylboronic acids were potent and selective for chymot-
rypsin-like proteasome activity and were cell-permeable. Ana-
logue 20 was one of the more active and selective of these
inhibitors and was also orally bioavailable in rodents. This
compound has demonstrated in vivo efficacy versus bortezomib
in several systemic human multiple myeloma xenograft models
and a similar to superior toxicity profile in mice and rats.25 An
orally administered treatment for patients with relapsed and
refractory multiple myeloma could represent a significant
advance in the field of proteasome inhibitor therapy in oncology.
On the basis of its in vitro potency, in vivo anticancer activities
(rodent), and druglike properties, 20 has been selected for
additional preclinical profiling for multiple myeloma therapy.
[(1R)-1-[[(2S,3R)-3-Hydroxy-2-[(6-phenylpyridine-2-carbon-
yl)amino]-1-oxobutyl]amino]-3-methylbutyl]boronic Acid (20).
A solution of N-[(1S,2R)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-
3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-meth-
ylbutyl]amino]carbonyl]-2-hydroxypropyl]-6-phenyl-2-pyridinecar-
boxamide (240 mg, 0.438 mmol), 2-methylpropylboronic acid (112
mg, 1.095 mmol), and 2 N aqueous HCl (0.44 mL) in a
heterogeneous mixture of methanol (3 mL) and hexane (3 mL) was
stirred at room temperature for 16 h. The methanolic layer was
removed, and the hexane layer was extract with methanol (5 mL).
The combined methanolic layers were combined and concentrated,
and the resulting residue was dissolved in EtOAc (30 mL). This
was washed with 8% aqueous NaHCO3, and the basic layers were
combined and extracted with EtOAc. The combined organic phases
were dried over sodium sulfate, concentrated in vacuo, and
chromatographed on silica gel using EtOAc/hexane gradient (50%)
followed by increasing amounts of methanol to elute 94 mg (52%)
of 20 as an off-white solid. HPLC indicates a purity of 97.6 area
Experimental Section
Carbamic Acid 1,1-Dimethylethyl Ester, N-[(1S,2R)-1-[[[(1R)-1-
[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-trimethyl-4,6-meth-
ano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-
2-hydroxypropyl] (11). To a cooled solution (0 °C) of Boc-L-
threonine (9.6 g, 43.76 mmol) dissolved in anhydrous DMF (60
mL) was added TBTU (14.06 g, 43.76 mmol), NMM (13.2 mL,
120 mmol), and the known 10 (12 g, 39.8 mmol). The mixture
stirred at room temperature for 16 h, poured into water, and
extracted with EtOAc (3 × 200 mL). Combined organics were
washed with 2% citric acid, 2% NaHCO3, and brine, dried over
anhydrous MgSO4, filtered, and evaporated to provide crude
product. Chromatography on silica gel using EtOAc/hexane gradient
(from 20% to 75%) afforded 13.1 g (71%) of 11 as a glassy solid:
1
%. H NMR (CD3OD, 400 MHz) δ 8.17 (m, 2H), 8.13 (m, 1H),
8.05 (m, 2H), 7.5 (m, 3H), 4.75 (d, J ) 3.04 Hz, 1H), 4.42 (dq, J
) 6.4, 2.92 Hz, 1H), 2.77 (t, b, 1H), 1.61 (m, 1H), 1.35 (t, J )
7.48 Hz, 2H), 1.29 (d, J ) 6.36 Hz, 3H), 0.89 (d, J ) 6.52 Hz,
6H); 13C NMR (CD3OD) δ 20.76, 22.64, 23.78, 27.17, 41.14, 57.19,
68.13, 121.93, 124.95, 128.16, 130.04, 131.18, 139.48, 140.24,
150.05, 157.79, 167.23, 177.43; MS m/z 452 (M + K), 436 (M +
Na), 396 (M - OH), 378, 352, 264. HRMS (M + Na) Calcd:
435.2056. Found: 435.2057. Anal. Calcd for C21H28BN3O5: C,
61.03; H, 6.83; N, 10.17%. Found: C, 63.22; H, 6.52; N, 10.17%.
1
mp 25°-30 °C; H NMR (DMSO-d6) δ 8.88 (br, 1H), 6.49 (d, J
) 8.4 Hz, 1H), 4.88 (d, J ) 5.8 Hz, 1H), 4.05 (m, 1H), 3.93 (m,
1H), 2.89 (m, 1H), 2.51 (m, 1H), 2.19 (m, 1H), 2.01 (m, 1H), 1.83
(t, J ) 5.9 Hz, 1H), 1.78 (m, 1H), 1.68 (m, 1H), 1.62 (m, 1H),
1.39 (s, 9H), 1.34 (d, J ) 10.0 Hz, 1H), 1.24 (s, 3H), 1.22 (s, 3H),
1.06 (d, J ) 6.4 Hz, 3H), 0.85 (d, J ) 6.4 Hz, 6H), 0.80 (s, 3H).
(2S,3R)-2-Amino-3-hydroxybutanamide, N-[(1R)-1-[(3aS,4S,-
6S,7aR)-Hexahydro-3a,5,5-trimethyl-4,6-methano-
1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]-, Hydrochloride Salt
(12). To a solution of 11 (13.1 g, 28.1 mmol) dissolved in anhydrous
diethyl ether (40 mL) at 0 °C was added 2 N HCl in diethyl ether
(98 mL, 196 mmol). The mixture was stirred overnight and slowly
allowed to warm to room temperature. The resulting white solid
was collected by filtration, washed, and dried under vacuum to
afford 7.9 g (70% yield) of 12 as a white solid. A second, less
pure crop was obtained from the mother liquors by concentration
to dryness (3.0 g, 26% yield). 1H NMR (DMSO-d6) δ 8.62 (d, J )
5.0 Hz, 1H), 8.17 (d, J ) 3.5 Hz, 3H), 4.28 (dd, J ) 8.8, 1.8 Hz,
1H), 3.78 (m, 1H), 3.52 (m, 1H), 3.00 (m, 1H), 2.28 (m, 1H), 2.10
(m, 1H), 1.92 (t, J ) 5.7 Hz, 1H), 1.84 (m, 1H), 1.75–1.62 (m,
2H), 1.43 (m, 1H), 1.31 (s, 3H,), 1.25 (s, 3H), 1.22 (d, J ) 10.6
Hz, 1H), 1.14 (d, J ) 6.2 Hz, 3H), 0.88 (d, J ) 6.4 Hz, 3H), 0.86
(d, J ) 6.4 Hz, 3H), 0.81 (s, 3H).
N-[(1S,2R)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-
trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methyl-
butyl]amino]carbonyl]-2-hydroxypropyl]-6-phenyl-2-pyridine-
carboxamide. To a solution of commercially available 6-phenylpy-
ridine-2-carboxylic acid (220 mg, 1.10 mmol) dissolved in DMF
(15 mL) and cooled to 0 °C were added TBTU (400 mg, 1.2 mmol),
NMM (0.35 mL, 3.2 mmol), and 12 (430 mg, 1.067 mmol). The
mixture was stirred for 2 h, poured in water, and extracted with
EtOAc. The combined organic layer was washed with 2% citric
acid, 2% NaHCO3, and brine, dried over MgSO4, filtered, and
Acknowledgment. We thank Drs. Jeffry Vaught and James
C. Kauer for their support and encouragement and acknowledge
the efforts of Dr. Renee Roemmele and Process Chemistry for
the large scale preparation of 20.
Note Added after Print Publication. To correct a printing error
related to Scheme 1, this paper was reposted on March 14, 2008.
Supporting Information Available: 1H NMR, HRMS, and
elemental analysis results for 7–10, 15–19, and 21–25 and details
of biological assay conditions. This material is available free of
charge via the Internet at http://pubs.acs.org.
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