Proteasome inhibitors for cancer therapy

Article abstract of DOI:10.1016/j.bmc.2012.02.007

Proteasome, a large multicatalytic proteinase complex that plays an important role in processing of proteins, has been shown to possess multiple catalytic activities. Among its various activities, the 'chymotrypsin-like' activity of proteasome has emerged as the focus of drug discovery efforts in cancer therapy. Herein we report chiral boronate derived novel, potent, selective and cell-permeable peptidomimetic inhibitors 6 and 7 that displayed activity against various rodent and human tumor cell lines (in vitro).

Full text of DOI:10.1016/j.bmc.2012.02.007

Bioorganic & Medicinal Chemistry 20 (2012) 2362–2368
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Proteasome inhibitors for cancer therapy
Mohamed Iqbal, Patricia A. Messina McLaughlin, Derek Dunn, Satish Mallya, Jean Husten,
⇑
Mark A. Ator, Sankar Chatterjee
Cephalon Inc., 145 Brandywine Parkway, West Chester, PA 19380, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteasome, a large multicatalytic proteinase complex that plays an important role in processing of pro-
teins, has been shown to possess multiple catalytic activities. Among its various activities, the ‘chymo-
trypsin-like’ activity of proteasome has emerged as the focus of drug discovery efforts in cancer
therapy. Herein we report chiral boronate derived novel, potent, selective and cell-permeable peptidom-
imetic inhibitors 6 and 7 that displayed activity against various rodent and human tumor cell lines
(in vitro).
Received 10 December 2011
Revised 27 January 2012
Accepted 2 February 2012
Available online 11 February 2012
Keywords:
Proteasome
Cancer
Ó 2012 Elsevier Ltd. All rights reserved.
Peptidomimetic
1. Introduction
chymotryptic, tryptic and peptidylglutamyl-like activities; thus
the complex is capable of cleaving most peptide bonds. However,
Proteasome is a large multicatalytic proteinase complex that
plays an important role in several critical cellular functions includ-
ing the processing of proteins involved in cell cycle progression
and gene expression.1,2 The 26S form of the proteasome, responsi-
ble for the ATP-dependent degradation of poly-ubiquitinated
proteins, is composed of one copy of the catalytic core, known as
the 20S subunit and two copies of the 19S regulatory subunit. The
X-ray crystal structure of the 20S subunit from an archaebacterial
proteasome revealed it to be a barrel-shaped structure made up of
four stacked rings.3 The two outer rings are each composed of
among its various activities, the ‘chymotrypsin-like’ activity of
the proteasome has emerged as the biological function of greatest
interest and the focus of drug discovery efforts. Increased levels of
this enzyme and subsequent protein breakdown have been
implicated in many disease states including muscular dystrophy,
cachexia accompanying cancer and malnutrition, lupus and can-
cers such as acute leukemia.5 The proteasome also controls levels
of proteins critical for cell cycle control, including p53, p27 and cy-
clin B, and is responsible for activation of the transcription factor
NF-jB through the degradation of its regulatory subunit IjB .
a 6
seven identical
a
-subunits and the two inner rings are each com-
Thus, development of the proteasome inhibitors has emerged as
an attractive target for cancer therapy.7 Previously, we reported
on novel, potent and selective peptidomimetic inhibitors of the
chymotrypsin-like activity of proteasome containing an aldehyde
enzyme reactive group at the P1-position (Fig. 1, compounds 1
and 2, IC50 values of 2 and 6 nM, respectively).8,9 Compound 1,
subsequently, found utility as a tool in proteasome research.10–14
posed of seven identical b-subunits. The catalytic nucleophile is
the hydroxyl group of the N-terminal threonine (Thr) of the b-sub-
unit; thus this enzyme is the first reported member of the threo-
nine proteinase class. The proteasome from higher organisms has
the same quaternary structure;4 however the seven
a and seven
b subunits are distinct from one another. The 20S proteasome
has been shown to possess multiple catalytic activities, including
We also communicated the corresponding a-ketocarboxamide
(3, IC50 13 nM),15 P0-extended ketoamide (4, IC50 1 nM)16 and
pinacol-derived boronate ester (5, IC50 6 nM)15 as potent inhibitors
of the chymotrypsin-like activity of proteasome (Fig. 1).
Abbreviations: BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; DMSO, dimethyl sulfoxide; HMPA, hexamethyl phosphora-
mide; HOBt, 1-hydroxybenzotriazole; NMM, N-methylmorpholine; DMF, N,N-
dimethyformamide; LDA, lithium diisopropylamide; THF, tetrahydrofuran; TBTU,
N,N,N0,N0-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate; HATU, 1-
hydroxy-7-azabenzotriazole uranium salt; HOAt, 1-hydroxy-7-azabenzotriazole;
AMC, 7-amido-4-methylcoumarin; AFC, 7-amido-4-trifluoromethylcoumarin; SDS,
sodium dodecyl sulfate; Tris, tris(hydroxymethyl)aminomethane; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid.
As a part of our continuous search for novel, potent, selective,
and drug-like inhibitors of the proteasome, we explored additional
enzyme reactive groups known to inhibit different classes of serine
and cysteine proteases (unpublished). We also employed a chiral
boronate derived from (+)-pinanediol as the enzyme reactive
group. This research resulted in potent, selective and cell-
permeable boronic ester inhibitors 6 and 7 (Fig. 2) that were active
in vitro against various tumor cell lines (rodent and human).
⇑
Corresponding author.
E-mail address: Sankar.Chatterjee24@gmail.com (S. Chatterjee).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.007

M. Iqbal et al. / Bioorg. Med. Chem. 20 (2012) 2362–2368
2363
O
1) R1 = CHO, R2 = N-Phthalimido
2) R1 = CHO, R2 = CN
H
N
R1
NO2
NH2
3) R1 = COCONHEt, R2 = CN
N
H
R2 (CH2)8
4) R1 = COCONH(CH2)2NHSO2Ph, R2 = CN
O
N
O
B
O
R2 = CN
5) R1 =
N
H
Figure 1. Chemical structures of compounds 1–5.
Pseudo P3
P2
P1
O
H
O
B
N
N
H
R1 (CH2)8
O
O
O
NO2
N
OH
N
N
N
B
N
NH2
OH
O
H
HO
6) R1 = CN
8
7) R1 = N-phthalimide
Figure 2. Chemical structures of compounds 6–8.
Subsequently, compounds 6and 7 provided the basis for additional
synthetic exploration en route to compound 8, a current clinical
candidate in the oncology area.17 In this report, we describe the
characteristics of compounds 6 and 7 disclosing their proteasome
inhibition data (against both isolated and intracellular enzyme)
and selectivity profile. We also disclose their in vitro activity
against a spectrum of tumor cell-lines.
While our effort was in progress, compound 9 (bortezomib,
Fig. 3) was approved for the treatment of multiple myeloma and
mantle cell lymphoma, validating the concept of targeted protea-
some inhibition in cancer therapy.18,19
20. Treatment of compound 20 with lithium hexamethyldisilazide
produced compound 21 that was desilylated in presence of hydro-
chloric acid to generate the free amine as its hydrochloride salt 22.
The procedure was adapted from various reports from Matteson’s
laboratories.20–22
Scheme 3 displays the synthesis of compounds 6, 7, 26 and 27
respectively. Commercially available tBoc-Arg (NO2)-OH (com-
pound 23) and boronate 22 (from Scheme 2) were coupled to af-
ford compound 24. Acidic treatment of compound 24 generated
compound 25 as its hydrochloride salt that was combined with
compounds 15 and 16 (from Scheme 1) to generate compounds 6
and 7, respectively. Compounds 6 and 7 were subsequently con-
verted to their corresponding boronic acid derivatives 26 and 27
via a boronate exchange reaction. All compounds were profiled
as mixtures of diastereomers at pseudo-P3 site (Fig. 2).
2. Chemistry
Syntheses of racemic acids 15 and 16 are shown in Scheme 1.
Commercially available cyclopentyl acetic acid (compound 10)
was esterified with benzyl alcohol to generate compound 11 that
was alkylated with 1,8-diiodooctane to produce compound 12. Dis-
placement of the iodo group in compound 12 with cyano and N-
phthalimido groups generated compounds 13 and 14, respectively.
Debenzylation of compounds 13 and 14 produced acids 15 and 16.
Scheme 2 depicts the synthesis of compound 22. Commercially
available boronic acid 17 was combined with (1S,2S,3R,5S)-(+)-
pinanediol (compound 18) to generate compound 19 that under-
went a stereospecific chlorination reaction to generate compound
3. Biology
3.1. Enzyme inhibition
Partial purification of the proteasome from postmortem human
liver was achieved by ion-exchange chromatography, ammonium
sulfate precipitation and gel filtration according to published proce-
dures.23,24 Compounds were tested for their ability to inhibit the
chymotrypsin-like (CT-like) activity of the 20S proteasome utilizing
the fluorogenic substrate succinyl-Leu-Leu-Val-Tyr-aminomethyl
coumarin (Suc-LLVY-AMC).25 They were also evaluated for their
ability to inhibit intracellular proteasome activity in Molt-4 human
T-cell leukemia lymphocytes.10 The ratio of IC50 values for inhibi-
tion of enzyme in Molt-4 cells vs. isolated enzyme was calculated
to offer a measure of the cell-permeability of an inhibitor (Table
1); a lower ratio reflected superior cell-permeability of an inhibitor.
Compounds were also profiled against several related enzymes, for
N
O
H
N
OH
N
N
H
B
O
OH
9
example, the proteasome’s trypsin-like activity, bovine
a-chymo-
trypsin ( -CT) and human calpain I, a cysteine protease (Table 1).
a
Figure 3. Chemical structure of compound 9.

2364
M. Iqbal et al. / Bioorg. Med. Chem. 20 (2012) 2362–2368
Y(CH2)8
COOZ Y(CH2)8
COOH
CH2COOX
e
b, c, d
12) Y = I, Z = CH2Ph
13) Y = CN, Z = CH2Ph
15) Y = CN
10) X = H
11) X = CH2Ph
a
16) Y = N-phthalimide
14) Y = N-phthalimde, Z = CH2Ph
Scheme 1. Reagents and conditions: (a) Benzyl alcohol, p-toluenesulfonic acid, benzene, reflux (Dean–Stark), 2 h, quantitative; (b) compound 11, LDA, 1,8-diiodooctane, THF–
hexane, HMPA, ꢀ78 to 0 °C, 2.5 h; 45%; (c) for compound 13: compound 12, NaCN, DMSO, 70–75 °C; 80%; (d) for compound 14: compound 12, potassium phthalimide, DMF,
70–75 °C; 85%; (e) (i) for compound 15: compound 13, H2, 10% Pd-C (50% water content); MeOH, 40 psi, 2 h, quantitative; (ii) for compound 16: compound 14, H2, 10% Pd-C
(50% water content); MeOH, 40 psi, 2 h, quantitative.
O
O
HO
OH
Cl
B
O
B
B
X
O
HO
B
O
b
c, d
a
O
+
HO
21) X = N(SiMe3)2
22) X = NH2.HCl
20
19
18
17
Scheme 2. Reagents and conditions: (a) Ether, room temp, 24 h; 94%; (b) (i) LDA in hexane–THF, compound 19 in THF–CH2Cl2, ꢀ70 °C, 1 h; (ii) 1.0 M ZnCl2 in ether, ꢀ78 °C,
3 h; 89%; (c) for compound 21: compound 20, LiN(SiMe3)2, THF, ꢀ78 °C to room temperature, overnight; quantitative; (d) for compound 22: compound 21, 4 N HCl in dioxane,
ether, 0 °C to room temperature 4 h; 73%.
O
O
O
XHN
O
tBocHN
COOH
N
B
O
H
N
B
Y(CH2)8
N
a, b
c,d
N
NO2
NH2
N
NO2
NH2
O
O
N
NO2
N
H
N
H
N
H
NH2
24) X = tBoc
23
6) Y = CN
25) X = H . HCl
e
7) Y = N-Phthalimide
O
H
N
OH
OH
26) Y = CN
Y(CH2)8
O
N
B
27) Y = N-Phthalimide
N
NO2
N
H
NH2
Scheme 3. Reagents and conditions: (a) for compound 24: compound 22 (Scheme 2), HATU, HOAt, NMM, DMF, 0 °C to room temperature, 5 h, 66% (b) for compound 25:
compound 24, ether–dioxane, 4 N HCl in dioxane, 0 °C to room temperature, 4 h, 90%; (c) for compound 6: compound 15 (Scheme 1), BOP, HOBt, NMM, DMF, 0 °C to room
temp, 60%; (d) for compound 7: compound 16 (Scheme 1), BOP, HOBt, NMM, DMF, 0 °C to room temp, 65%; (e) 2-methylpropylboronic acid, 2 N HCl, MeOH, hexane, 70–75%.
3.2. In vitro anti-tumor activity
4. Discussion
The effects of the inhibitors 6 and 7 on in vitro viability of a pa-
nel of human and rodent cell lines representing different tumor
types were investigated. The cell lines were AT-2 (rat prostatic car-
cinoma), DU-145 (human prostatic carcinoma), PANC-1 (human
pancreatic carcinoma), SKMEL-5 (human melanoma), OVCAR-3
(human ovarian carcinoma), MCF-7 (human breast cell carcinoma),
and Lewis lung (mouse lung carcinoma), respectively. The results
of these studies are presented in Table 2.
As shown in Table 1, both boronic esters 6 and 7 and boronic
acids 26 and 27 were potent inhibitors of the chymotrypsin-like
activity of isolated proteasome (IC50 ca. 4–9 nM). These com-
pounds were uniformly unable to inhibit the trypsin-like activity
of the enzyme complex at concentrations up to 10
-chymotrypsin-like activity (IC50 >10 M), and human calpain I,
a cysteine protease at concentrations up to 10 M, respectively.
However, the boronic esters 6 and 7 displayed superior potency
lM, bovine
a
l
l

M. Iqbal et al. / Bioorg. Med. Chem. 20 (2012) 2362–2368
2365
Table 1
Enzyme inhibition data for compounds 6, 7, 26 and 27
Compd
Prot. CT-like
IC50 (nM)
Prot. CT-like Molt-4
IC50 (nM)
Ratio Molt-4
IC50/enzyme IC50
Prot. trypsin-like
Bovine
% inh @ 10
a
-CT
l
Human calpain
Mb
Mb
I % inh @ 10 l
Mb
a
a
% inh @ 10
l
6
7
26
27
3.5 0.5
3.5 1.3
8.7 3.2
4.1 1.6
240 50
70 10
7340 1640
2830 1560
69
20
840
690
0
20
5
24
30
26
30
2
11
0
3
0
a
Mean S.D., n = 3–6.
b
Results are the averages of two independent experiments, each performed in triplicate.
Table 2
Effect of compounds 6 and 7 on viability of tumor cell linesa
Compd
AT-2 IC50 (nM)
DU-145 IC50 (nM)
PANC-1 IC50 (nM)
SKMEL-5 IC50 (nM)
OVCAR-3 IC50 (nM)
MCF-7 IC50 (nM)
Lewis lung IC50 (nM)
6
7
>1
>1
l
l
M
M
293
167
283
184
85
82
69
29
425
292
585
513
a
Results are the averages of two independent experiments, each performed in triplicate.
in the Molt-4 cell assay (IC50 240 and 70 nM, respectively) com-
pared to the corresponding boronic acids 26 and 27 (IC50 7340
and 2830 nM, respectively). This was reflective of the superior
cell-permeability of compounds 6 and 7. However, it should be
noted that both bortezomib (compound 9) and compound 8, next
generation inhibitor from follow-on work from our laboratories be-
long to the class of boronic acids. However, based on their superior
cell activity, compounds 6 and 7 were advanced and profiled
against a wide range of tumor types as shown in Table 2. Both com-
pounds displayed significant activity against various tumor cell-
lines except AT-2 (rat prostatic tumor) in the range of inhibitor
concentrations tested, but were efficacious against the human
prostate tumor line DU-145. The basis for this difference is un-
known at this time. Based on their in vitro potency, intra-cellular
inhibitory capability and in vitro anti-tumor activity, compounds
6 and 7 had emerged as the launching pad for additional explora-
tion/modification in the series.
6.1.2. Benzyl-2-cyclopentyl acetate (11)
A mixture of cyclopentylacetic acid (compound 10, 10.02 g,
78.2 mmol), benzyl alcohol (8.45 g, 78.2 mmol) and p-toluenesul-
fonic acid monohydrate (1.48 g, 7.82 mmol) in benzene (60 mL)
was refluxed under Dean–Stark condition for 2 h, cooled, concen-
trated under vacuum and redissolved in ether (60 mL). The organic
layer was washed successively with water (1 ꢁ 25 mL), saturated
aq NaHCO3 solution (2 ꢁ 25 mL), water (1 ꢁ 25 mL), and brine
(1 ꢁ 25 mL), dried (MgSO4) and evaporated to generate compound
11 (15.40 g, quantitative) as a viscous oil that was utilized in next
step without further purification. 1H NMR (300 MHz, CDCl3) d: 7.35
(m, 5H), 5.10 (s, 2H), 2.40 (d, J = 8 Hz, 2H), 2.26 (m, 1H), 1.81 (m,
2H), 1.6 (m, 4H), 1.18 (m, 2H).
6.1.3. 10-Iodo-2-[(R,S)-cyclopentyl]decanoic acid, benzyl ester
(12)
A solution of compound 11 (3.96 g, 18 mmol) in anhydrous THF
(10 mL) was added dropwise to a freshly prepared solution of lith-
ium diisopropylamide (20 mmol) in hexane/THF (8 mL/20 mL)
maintained at ꢀ78 °C. The mixture was stirred for additional
0.5 h and to it a solution of 1,8-diiodooctane (7.19 g, 20 mmol) in
hexamethylphosphoramide (3.50 g, 20 mmol) was added. The
reaction mixture was stirred at ꢀ78 °C for 0.5 h, slowly brought
to 0 °C over a period of 2 h, quenched by slow addition of 12% aq
NH4Cl (50 mL) and extracted with ether (3 ꢁ 50 mL). The com-
bined organic layer was washed with brine (2 ꢁ 25 mL), dried
(MgSO4) and concentrated to generate a crude product that was
purified by flash chromatography (silica gel, 1% ethyl acetate in
hexanes) to generate compound 12 (3.23 g, 45%) as an oil; 1H
NMR (300 MHz, CDCl3) d: 7.35 (m, 5H), 5.15 (s, 2H), 3.20 (t,
J = 8 Hz, 2H), 2.20 (m, 1H), 2.00 (m, 1H), 1.80–1.20 (a series of m,
22H).
5. Conclusion
In this report, we disclosed compounds 6 and 7, chiral boronate
derived potent, selective and cell-permeable peptidomimetic
inhibitors of the chymotrypsin-like activity of proteasome. These
inhibitors displayed activity against various rodent and human tu-
mor cell lines (in vitro). This research provided the impetus for
additional exploration that led to potent, selective and orally bio-
available (rat and mouse) inhibitor 8 that entered into clinic for
the treatment of myeloma.
6. Experimentals
6.1. Chemistry
6.1.4. 10-Cyano-2-[(R,S)-cyclopentyl]decanoic acid, benzyl ester
(13)
6.1.1. General
Commercially available reagents and solvents were utilized
without any further purification. 1H NMR spectra were obtained
at 300 or 400 MHz in the solvent indicated with tetramethylsilane
(TMS) as an internal standard. Coupling constants (J) are reported
in Hertz (Hz). The following abbreviations were used: s: singlet;
d: doublet; dd: doublet of doublet; m: multiplet; q: quartet; t:
triplet and b: broad. Column chromatography was performed on
230–400 mesh silica gel 60. Analytical reverse phase HPLC was
performed using a Zorbax RX-C8, 5 ꢁ 150 mm column eluting with
a mixture of acetonitrile and water containing 0.1% trifluoroacetic
acid with a gradient of 10–100%.
A mixture of compound 12 (3.99 g, 8.7 mmol) and sodium
cyanide (0.47 g, 9.6 mmol) in anhydrous DMSO (15 mL) was heated
at 70–75 °C in a well-ventilated hood for 0.5 h, cooled and
quenched with ice-water (ꢂ50 g). The aqueous layer was extracted
into ether (3 ꢁ 25 mL) and the combined organic layer was succes-
sively washed with water (1 ꢁ 25 mL) and brine (1 ꢁ 25 mL), dried
(MgSO4) and evaporated to generate compound 13 (2.89 g, 80%) as
a
viscous oil that was utilized without further purification.
1H NMR (300 MHz, CDCl3) d: 7.35 (m, 5H), 5.15 (s, 2H), 2.30
(t, J = 8 Hz, 2H), 2.20 (m, 1H), 2.00 (m, 1H), 1.80–1.30 (m, 22H).

2366
M. Iqbal et al. / Bioorg. Med. Chem. 20 (2012) 2362–2368
6.1.5. 10-(N-Phthalimido)-2-[(R,S)-cyclopentyl]decanoic acid,
benzyl ester (14)
1.09 (d, J = 10.1 Hz, 1H); 0.9 (d, J = 6.8 Hz, 3H); 0.87 (d, J = 6.4 Hz,
3H); 0.82 (s, 3H).
A mixture of compound 12 (1.21 g, 2.6 mmol) and potassium
phthalimide (0.54 g, 3.0 mmol) in anhydrous DMF (8 mL) was
heated at 70–75 °C for 0.5 h, cooled and quenched with ice-water
(ꢂ40 g). The aqueous layer was extracted into ether (3 ꢁ 25 mL)
and the combined organic layer was successively washed with
water (1 ꢁ 25 mL) and brine (1 ꢁ 25 mL), dried (MgSO4) and
evaporated to generate compound 14 (1.24 g, 85%) as a viscous
oil that was utilized directly in the next step. 1H NMR
(300 MHz, CDCl3) d: 7.85 (m, 2H), 7.70 (m, 2H), 7.35 (m, 5H),
5.15 (s, 2H), 2.30 (t, J = 8 Hz, 2H), 2.20 (m, 1H), 2.00 (m, 1H),
1.80–1.30 (m, 22H).
6.1.10. N,N-Bis(trimethylsilyl)-(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-
3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-
methylbutylamine (21)
A solution of lithium bis(trimethylsilyl)amide in THF (1.0 M,
189 mL, 0.189 mol) was added dropwise to a solution of compound
20 (59.0 g, 0.189 mol) in anhydrous THF (580 mL) maintained at
ꢀ78 °C. The reaction mixture was allowed slowly to return to room
temperature, stirred overnight and concentrated. The crude resi-
due was taken into dry hexane (800 mL), stirred for 2 h and filtered
through a celite pad. Removal of solvent produced compound 21
(79 g, quantitative) as an oil that was immediately taken into the
next step. 1H NMR (300 MHz, DMSO-d6) d: 4.33 (dd, J = 8.6 Hz,
1.5 Hz, 1H); 2.58 (m, 1H); 2.29 (m, 1H); 2.18 (m, 1H); 1.95 (t,
J = 5.9 Hz, 1H); 1.85 (m, 1H); 1.9–1.55 (m, 3H); 1.31 (s, 3H); 1.24
(s, 3H); 1.17 (m, 1H); 1.01 (d, J = 10.6 Hz, 1H); 0.85 (d, J = 6.6 Hz,
3H), 0.83 (d, J = 6.6 Hz, 3H); 0.80 (s, 3H); 0.08 (s, 18H).
6.1.6. 10-Cyano-2-[(R,S)-cyclopentyl]decanoic acid (15)
A mixture of compound 13 (2.88 g, 8.00 mmol) and 10% Pd-C
(0.6 g, DeGussa, 50% H2O content) in anhydrous methanol
(35 mL) was hydrogenated for 2 h (42–26 psi), filtered through a
pad of celite and concentrated to generate 2.02 g (quantitative)
of compound 15 as a viscous oil. 1H NMR (300 MHz, CDCl3) d:
2.35 (t, J = 8 Hz, 2H), 2.20 (m, 1H), 2.00 (m, 1H), 1.90–1.30 (a series
of m and a broad, 23H).
6.1.11. (1R)-1-[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-trimethyl-4,6-
methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutylamine
hydrochloride salt (22)
A solution of 4 N HCl in dioxane (193 mL, 0.772 mol) was slowly
added to a solution of compound 21 (79.00 g, 0.193 mol) in diox-
ane (100 mL) and ether (200 mL) maintained at 0 °C. The cooling
bath was removed and the reaction mixture was stirred for 4 h at
room temperature and concentrated. The residue was taken into
anhydrous hexane (500 mL), cooled to 0 °C and treated with 2 M
solution of HCl in diethyl ether (48 mL, 0.096 mol). The reaction
mixture was stirred at 0 °C for 1 h, concentrated, diluted with
anhydrous hexane and stirred at room temperature overnight.
The solid that separated was filtered and dried under high vacuum
to afford compound 22 (38.1 g, 73%). An additional crop of 4.13 g
was obtained on concentration of the mother liquor. 1H NMR
(300 MHz, DMSO-d6) d 7.85 (br, 3H), 4.45 (t, J 9.2 Hz, 1H), 2.78
(m, 1H), 2.34 (m, 1H), 2.21 (m, 1H), 2.01 (t, J = 5.3 Hz, 1H), 1.89
(m, 1H), 1.82–1.65 (m, 3H), 1.49 (m, 1H), 1.38 (s, 3H), 1.27
(s, 3H), 1.12 (d, J = 1.1 Hz, 1H), 0.87 (d, J = 6.6 Hz, 6H), 0.83 (s, 3H).
6.1.7. 10-(N-Phthalimido)-2-[(R,S)-cyclopentyl]decanoic acid (16)
Following the same procedure as described above for the syn-
thesis of compound 15, 0.41 g of compound 14 was hydrogenated
to afford compound 16 as viscous oil (0.31 g, quantitative). 1H NMR
(300 MHz, CDCl3) d: 7.85 (m, 2H), 7.70 (m, 2H), 3.70 (t, J = 8.0 Hz,
2H), 2.15 (m, 1H), 1.95 (m, 1H), 1.85–1.10 (a series of m and a
broad, 23H).
6.1.8. (2-(2-Methylpropyl)-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-
trimethyl-4,6-methano-1,3,2-benzodioxaborole (19)
A mixture of 2-methylpropylboronic acid (compound 17,15.00 g,
0.147 mol) and (+)-pinanediol (compound 18, 23.90 g, 0,140 mol) in
ether (300 mL) was stirred at room temperature for 24 h, dried
(Na2SO4) and evaporated to dryness. The crude residue was purified
by flash column chromatography (silica gel, hexane–ethyl acetate:
9:1) to generate compound 19 as an oil (32.60 g, 94%). 1H NMR
(300 MHz, DMSO-d6) d: 4.28 (dd, J = 8.8 Hz, 2.0 Hz, 1H); 2.30
(m, 1H); 2.18 (m, 1H); 1.96 (t, J = 5.3 Hz, 1H); 1.86 (m, 1H); 1.78 (d,
J = 6.8 Hz, 1H); 1.68 (m, 1H); 1.30 (s, 3H); 1.25 (s, 3H); 1.01 (d,
1H); 0.9 (d, J = 6.6 Hz, 6H); 0.81 (s, 3H); 0.69 (m, 2H).
6.1.12. Carbamic acid 1,1-dimethylethyl ester, N-[(1S)-1-[[[(1R)-
1-[3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,
2-benzodioxoborol-2-yl]-3-methylbutyl]amino]carbonyl]-4-
[[imino(nitroamino)methyl]amino]butyl] (24)
A cooled (0 °C) solution of commercially available compound 23
(15.70 g, 49.30 mmol) in anhydrous DMF (100 mL) was succes-
sively treated with NMM (13.60 mL, 123 mmol), HOAt (6.71 g,
49.30 mmol) and HATU (18.70 g, 49.30 mmol). The mixture was
stirred for an additional 15 min and then treated with compound
22 (12.40 g, 41.10 mmol). The cooling bath was removed and the
mixture was stirred for 5 h, diluted with ethyl acetate (800 mL)
and washed successively with 2% aq citric acid solution
(2 ꢁ 150 mL), 2% aq NaHCO3 solution (2 ꢁ 150 mL) and brine
(2 ꢁ 100 mL). Each aqueous phase was re-extracted with ethyl ace-
tate (1 ꢁ 100 mL). The combined organic layer was dried (Na2SO4),
concentrated under high vacuum, dissolved in ether (150 mL) and
triturated with hexane (600 mL). The separated solid was filtered
and purified by flash column chromatography (silica gel, eluant:
1:1 ethyl acetate to ethyl acetate) to generate compound 24
(15.2 g, 66%); 1H NMR (300 MHz, DMSO-d6) d 8.80 (br, 1H), 8.50
(br, 1H), 7.87 (br, 2H), 7.01 (br, 1H), 4.07 (dd, J = 7.9 Hz, 2.01 Hz,
1H), 4.00 (m, 1H), 3.12 (m, 2H), 2.55 (m, 1H), 2.20 (m, 1H), 2,01
(m, 1H), 1.83 (t, J = 5.1 Hz, 1H), 1.78 (m, 1H), 1.74–1.44 (m, 6H),
1.38 (s, 9H), 1.33 (m, 1H), 1.25 (s, 2H), 1.24 (s, 3H), 1.22 (s, 3H),
0.84 (d, J = 6.6 Hz, 6H), 0.81 (s, 3H).
6.1.9. 2-[(1S)-1-Chloro-3-methylbutyl]-(3aS,4S,6S,7aR)-hexahy
dro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborole)
(20)
A freshly prepared cooled solution (ꢀ30 °C) of lithium diisopro-
pylamide (LDA, 0.254 mol) in hexane–THF (1:2, 100 mL) was
added slowly via canulation to a solution of compound 19 (50 g,
0.212 mol) in a mixture of THF (700 mL)–CH2Cl2 (50 mL), cooled
to ꢀ70 °C and maintained at that temperature throughout the
addition. After an additional stirring of 30 min, a solution of ZnCl2
(1.0 M in ether, 340 mL, 0.34 mol) was slowly added to the reaction
mixture that was stirred at ꢀ78 °C for an additional 3 h, slowly
brought to room temperature, evaporated to dryness and
partitioned between petroleum ether (1 L) and 10% aq NH4Cl
solution (800 mL). The separated aqueous layer was further
extracted into petroleum ether (2 ꢁ 150 mL). The combined
organic layers were dried (Na2SO4) and concentrated to generate
compound 20 as an oil (59 g, 89%) that was utilized without further
purification. 1H NMR (300 MHz, DMSO-d6) d: 4.43 (dd, J = 8.8 Hz,
1.8 Hz, 1H); 3.59 (m, 1H); 2.33 (m, 1H); 2.21 (m, 1H); 2.01 (m,
1H); 1.88 (m, 1H); 1.84–1.55 (m, 4.H); 1.34 (s, 3H); 1.26 (s, 3H);

M. Iqbal et al. / Bioorg. Med. Chem. 20 (2012) 2362–2368
2367
6.1.13. (2S)-2-Amino-5-[[imino(nitroamino)methyl]amino]
pentamide-N-[(1R)-1-[3aS,4S,6S,7aR)-hexahydro-3a,5,5-tri
methyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methyl butyl]-,
hydrochloride salt (25)
6.3.1. [(1R)-1-[[(2S-5-[[Imino(nitroamino)methyl]-2-[((R,S)-10-
cyano-2-cyclopentyldecanoyl)amino-1-oxopentyl]amino]-3-
methylbutyl]boronic acid (26)
MS m/e 562 (M+H–H2O). High resolution MS: calculated for
A solution of compound 24 (4.04 g, 7.06 mmol) in a mixture of
dioxane (40 mL) and ether (7 mL) maintained at 0 °C was treated
with a 4 N solution of HCl in dioxane (15 mL). The cooling bath
was removed and the reaction mixture was stirred for an addi-
tional 4 h, concentrated under high vacuum, and stirred in ether
(50 mL) overnight to generate compound 25 (3.18 g, 90%). 1H
NMR (300 MHz, DMSO-d6) d: 8.56 (br, 2H), 8.22 (br, 3H), 7.97 (br,
2H), 4.28 (dd, J = 8.6 Hz, 2.0 Hz, 1H), 3.77 (m, 1H), 3.04 (m, 2H),
2.28 (m, 1H), 2.11 (m, 2H), 1.92 (t, J = 5.5 Hz, 1H), 1.83 (m, 1H),
1.79–1.59 (m, 4H), 1.31 (s, 4H), 1,19 (d J = 6.0 Hz, 1H), 1.24
(s, 3H), 1.22 (s, 3H), 0.84 (d, J = 6.6 Hz, 6H), 0.81 (s, 3H)
C27H50BN7O6 562.3891 (M+H–H2O); found 562.3888 (M+H–H2O).
6.3.2. [(1R)-1-[[(2S-5-[[Imino(nitroamino)methyl]-2-[((R,S)-10-
N-phthalimido-2-cyclopentyldecanoyl)amino-1-oxopentyl]
amino]-3-methylbutyl]boronic acid (27)
MS m/e 682 (M+H–H2O). High resolution MS: calculated for
C34H54BN7O8 682.4103 (M+H–H2O); found 882.4100 (M+H–H2O).
6.4. Biology
6.4.1. Assay for the chymotrypsin-like activity of human liver
proteasome
20S proteasome isolated from human liver was diluted to a final
concentration of 6.7 lg/mL in 96-well U-bottom white plates (Dy-
6.2. Compounds 6 and 7
nex Technologies #6905) in buffer containing 20 mM Tris–Cl (pH
7.5), 0.04% SDS and 5% DMSO. Full activation of the enzyme was at-
tained by pre-incubating 15 min at 27 °C. Reactions were started
by addition of the fluorogenic substrate Succinyl-Leu-Leu-Val-
Tyr-aminomethyl coumarin (Suc-LLVY-AMC; Bachem I-1395) to a
General procedure: A cooled (0 °C) solution of compounds 15 or
16 (1 equiv) in anhydrous DMF was successively treated with
NMM (3 equiv), HOBt (1.1 equiv) and BOP (1.1 equiv). The mixture
was stirred for an additional 15 min and then treated with com-
pound 25 (1.05 equiv). The cooling bath was removed and the mix-
ture was stirred for 5 h, diluted with ethyl acetate and washed
successively with 2% aq citric acid solution, 2% aq NaHCO3 solution
and brine. Each aqueous phase was re-extracted with ethyl acetate.
Combined organic layer was dried (Na2SO4), concentrated under
high vacuum and purified by flash column chromatography (elu-
ant: methylene chloride–methanol in increasing concentration)
to generate compounds 6 or 7 (60–65%).
concentration of 100 lM. The 100 lL assays were incubated at
27 °C and the release of AMC monitored by the increase in fluores-
cence (kex 360 nm/kem 460 nm) every 1.5 min for 20 min (Cytoflu-
or). The rate of product formation was evaluated at each inhibitor
concentration. The IC50 values were calculated by non-linear
regression using GraphPad Prism.
6.4.2. Cellular assay for thechymotrypsin-like activity of protea-
some in Molt-4 cells
Protease inhibitors were tested for intracellular activity in Molt-
4 human T-cell leukemia lymphocytes (ATCC#CRL-1582). Cells
6.2.1. 2-[[(1R)-1-[[(2S-5-[[Imino(nitroamino)methyl]-2-[((R,S)-
10-cyano-2-cyclopentyldecanoyl)amino-1-oxopentyl]amino]-
3-methylbutyl]]-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,
6-methano-1,3,2-benzodioxaborole (6)
were washed and resuspended to
a final concentration of
6 ꢁ 106 cells/mL in 5.4 mM KCl, 120 mM NaCl, 25 mM glucose,
1.5 mM MgSO4, 1 mM Na pyruvate, 20 mM HEPES (pH 7.0) and dis-
pensed to 96-well U-bottom white plates. Cells were treated with
test compounds for 15 min at 37 °C prior to the addition of the
lipophillic substrate MeOSuc-Phe-Leu-Phe-AFC (Enzyme Systems
1H NMR (300 MHz, DMSO-d6) d: 8.50 (br, 2H), 8.00 (br, 3H), 4.40
(m, 1H), 4.15 (m, 1H), 3.20 (m, 2H), 2.70 (m, 1H), 2.50 (t, 2H), 2.20–
1.25 (a series of overlapping d, t and m for aliphatic hydrogens,
37H), 1.24 (s, 3H), 1.22 (s, 3H), 0.84 (d, J = 6.6 Hz, 6H), 0.81 (s,
3H). MS m/e 714 (M+H). High resolution MS: calculated for
Products, AFC-088) to a concentration of 100
lM at a final concen-
tration of 0.6% DMSO. The 100 L assays were incubated at 37 °C
l
C37H64BN7O6 714.5090 (M+H); found 714.5095 (M+H).
and the release of AFC monitored by the increase in fluorescence
(kex 420 nm/kem 490 nm) every 1.5 min for 20 min (Cytofluor).
Inhibition was evaluated as for the isolated enzyme.
6.2.2. 2-[[(1R)-1-[[(2S-5-[[Imino(nitroamino)methyl]-2-[((R,S)-
10-N-phthalimido-2-cyclopentyldecanoyl)amino-1-oxopentyl]
amino]-3-methylbutyl]]-(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trim
ethyl-4,6-methano-1,3,2-benzodioxaborole (7)
Acknowledgements
1H NMR (300 MHz, DMSO-d6) d: 8.50 (br, 2H), 8.00 (br, 3H), 7.85
(m, 2H), 7.70 (m, 2H), 4.40 (m, 1H), 4.15 (m, 1H), 3.20 (m, 2H), 2.70
(m, 1H), 2.50 (t, 2H), 2.20–1.25 (a series of overlapping d, t and m
for aliphatic hydrogens, 37H), 1.24 (s, 3H), 1.22 (s, 3H), 0.84 (d,
J = 6.6 Hz, 6H), 0.81 (s, 3H). MS m/e 834 (M+H). High resolution
MS: calculated for C44H68BN7O8 834.5307 (M+H); found 834.5300
(M+H).
Authors wish to acknowledge Drs. Jeffry Vaught, James C. Kauer
and John P. Mallamo for their keen interest, support and encour-
agement during the course of this research. We thank Dr. Bruce
Ruggeri for providing us with tumor cell line data. Authors sin-
cerely thank all the reviewers for their constructive criticism and
valuable suggestions to improve the quality of the manuscript.
References and notes
6.3. Compounds 26 and 27
1. For reviews on proteasome see (a) Weissman, A. M.; Shabek, N.; Ciechanover, A.
Nat. Rev. Mol. Cell Biol. 2011, 12, 605. Erratum in Nat. Rev. Mol. Cell Biol. 2011, 12,
686; (b) Lipkowitz, S.; Weismann, A. M. Nat. Rev. Cancer 2011, 11, 629; (c)
Borissenko, L.; Groll, M. Chem. Rev. 2007, 107, 687.
2. Nalepa, G.; Rolfe, M. Harper Nat. Rev. Drug. Discov. 2006, 5, 596.
3. Lowe, J.; Stock, D.; Jap, B.; Zwick, P.; Baumeister, W.; Huber, R. Science 1995,
268, 533.
4. Groll, M.; Ditzel, L.; Lowe, J.; Bochtler, M.; Bartunik, H.; Huber, R. Nature 1997,
386, 463.
5. (a) Abu-Baker, A.; Messaed, C.; Laganiere, J.; Gaspar, C.; Brais, B.; Rouleau, G. A.
Hum. Mol. Genet. 2003, 12, 2609; (b) Attaix, D.; Combaret, L.; Tilignac, T.;
General procedure: A mixture of compound 6 or 7 (1 equiv), 2-
methylpropylboronic acid (2.5 equiv) and 2 N aqueous hydrochlo-
ric acid in a heterogeneous mixture of methanol and hexane was
stirred overnight at room temperature, concentrated, diluted with
EtOAc, washed with saturated NaHCO3 solution and evaporated to
dryness. Purification of residue by column chromatography (elu-
ant: methylene chloride–methanol in increasing concentration)
yielded compound 26 or 27 (70–75%).

2368
M. Iqbal et al. / Bioorg. Med. Chem. 20 (2012) 2362–2368
Taillandier, D. Mol. Biol. Rep. 1999, 26, 77; (c) Chen, M.; von Mikecz, A.
15. Iqbal, M.; Chatterjee, S.; Kauer, J. C.; Mallamo, J. P.; Messina, P. A.; Reiboldt, A.;
Siman, R. Bioorg. Med. Chem. Lett. 1996, 6, 287.
16. Chatterjee, S.; Dunn, D.; Mallya, S.; Ator, M. A. Bioorg. Med. Chem. Lett. 1999, 9,
2603.
Autoimmun. Rev. 2005, 4, 117; (d) Kumatory, A.; Tanaka, K.; Inamura, N.; Sone,
S.; Ogura, T.; Matsumoto, T.; Tachikawa, T.; Shin, S.; Ichihara, A. Proc. Natl. Acad.
Sci. U.S.A. 1990, 87, 7071.
6. Rolfe, M.; Chiu, M. I.; Pagano, M. J. Mol. Med. 1997, 75, 5.
7. (a) Sippl, W.; Collura, V.; Colland, F. Future Oncol. 2011, 7, 619; (b) Mitsiades, C.
S.; Mitsiades, N.; Hideshima, T.; Richardson, P. G.; Anderson, K. C. Curr. Drug.
Targets 2006, 7, 1341; (c) Rajkumar, S. V.; Richardson, P. G.; Hideshima, T.;
Anderson, K. C. J. Clin. Oncol. 2005, 23, 630.
17. (a) www.clinicaltrials.gov (accessed on 08.12.11).; (b) Dorsey, B. D.; Iqbal, M.;
Chatterjee, S.; Menta, E.; Bernardini, R.; Bernareggi, A.; Cassara, P. G.; D’Arasmo,
G.; Ferretti, E.; De Munari, S.; Oliva, A.; Ambrogio, P.; Pezzoni, G.; Allievi, C.;
Strepponi, I.; Ruggeri, B.; Ator, M. A.; Williams, M.; Mallamo, J. P. J. Med. Chem.
2008, 51, 1068.
8. Iqbal, M.; Chatterjee, S.; Kauer, J. C.; Das, M.; Messina, P.; Freed, B.; Biazzo, W.;
Siman, R. J. Med. Chem. 1995, 38, 2276.
18. For a review on bortezomib, see Roccaro, A. M.; Hideshima, T.; Richardson, P.
G.; Russo, D.; Ribatti, D.; Vacca, A.; Dammacco, F.; Anderson, K. C. Curr. Pharm.
Biotechnol. 2006, 7, 441–448.
9. For subsite nomenclature for
a peptide-based inhibitor, see Schechter, I.;
Berger, A. Biochem. Biophys. Res. Commun. 1967, 118, 157.
10. Harding, C. V.; France, J.; Song, R.; Farah, J. M.; Chatterjee, S.; Iqbal, M.; Siman,
R. J. Immunol. 1995, 155, 1767.
11. An, B.; Goldfarb, R. H.; Siman, R.; Dou, Q. P. Cell Death Diff. 1998, 5, 1062.
12. Kitson, R. P.; Lu, M.; Siman, R.; Goldfarb, R. H. In Vivo 2000, 14, 265.
13. Sun, J.; Nam, S.; Lee, C.; Li, B.; Coppola, D.; Hamilton, A. D.; Dou, Q. P.; Sebti, S.
M. Cancer Res. 2001, 61, 1280.
19. For an excellent Perspective on development of bortezomib from bench to
bedside in record time, see Sanchez-Serrano, I. Nat. Rev. Drug Dis. 2006, 5, 107.
20. Matteson, D. S.; Majumdar, D. Organometallics 1983, 2, 1529.
21. Matteson, D. S.; Sadhu, K. M. Organometallics 1984, 3, 614.
22. Matteson, D. S. Tetrahedron 1859, 1989, 45. and references cited therein.
23. Driscroll, J.; Goldberg, A. L. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 787.
24. Yamamoto, T. M.; Nojima, S.; Ishiura, S.; Sugita, H. Biochim. Biophys. Acta 1986,
882, 297.
14. Kim, W.; Horbinski, C.; Sigurdson, W.; Higgins, D. J. Neurochem. 2004, 91,
1044.
25. Stein, R. L.; Melandri, F.; Dick, L. Biochemistry 1996, 35, 3899.