Optimization of a series of dipeptides with a P3 β-neopentyl asparagine residue as non-covalent inhibitors of the chymotrypsin-like activity of human 20S proteasome

Article abstract of DOI:10.1039/c2md20060k

Inhibition of the proteasome by covalent inhibitors is a clinically proven anti-cancer therapy. We report here that dipeptides with a P3 neopentyl Asn residue are potent, reversible, non-covalent inhibitors selective for the chymotryptic activity of the 20S proteasome in vitro and in cells. The X-ray structure of compound 20 in complex with yeast 20S reveals the importance of hydrophobic bonding interactions of the neopentyl group within the S3 binding pocket of the 20S β5 sub-unit. Four compounds show comparable potencies to boronic acid inhibitors in a panel of assays.

Full text of DOI:10.1039/c2md20060k

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CONCISE ARTICLE
Optimization of a series of dipeptides with a P3 b-neopentyl asparagine
residue as non-covalent inhibitors of the chymotrypsin-like activity of human
20S proteasome†
*
Christopher Blackburn, Cynthia Barrett, Jonathan L. Blank, Frank J. Bruzzese, Nancy Bump,
Lawrence R. Dick, Paul Fleming, Khristofer Garcia, Paul Hales, Matthew Jones, Jane X. Liu,
Masayuki Nagayoshi, Darshan S. Sappal, Michael D. Sintchak, Christopher Tsu, Cindy Xia, Xiansi Zhou
and Kenneth M. Gigstad
Received 29th February 2012, Accepted 16th April 2012
DOI: 10.1039/c2md20060k
Inhibition of the proteasome by covalent inhibitors is a clinically proven anti-cancer therapy. We report
here that dipeptides with a P3 neopentyl Asn residue are potent, reversible, non-covalent inhibitors
selective for the chymotryptic activity of the 20S proteasome in vitro and in cells. The X-ray structure of
compound 20 in complex with yeast 20S reveals the importance of hydrophobic bonding interactions of
the neopentyl group within the S3 binding pocket of the 20S b5 sub-unit. Four compounds show
comparable potencies to boronic acid inhibitors in a panel of assays.
crystallography.18 Two additional boronic acids MLN97088,19
and CEP-1877020,21 are undergoing clinical trials.
1. Introduction
The ubiquitin-proteasome pathway is responsible for the regu-
Non-covalent inhibitors of the catalytic b sub-units of the 20S
core particle are less well described even though they may show
involved in signal transduction, cell cycle control, apoptosis and
more widespread tissue distribution than their covalent coun-
lated degradation of intracellular proteins, including those
inflammation.1–3 Inhibition of the 20S core particle of the pro-
terparts due to rapid dissociation from the 20S sub-unit in the
teasome causes accumulation of substrate proteins leading to
blood compartment. Among the non-covalent inhibitors repor-
apoptosis and is preferentially cytotoxic to cancer cells.4,5 The
ted, cyclic peptide TMC-95A, competitively inhibits the
active sites of 20S located on the b1, b2, and b5 subunits (clas-
chymotrypsin-like activity of the proteasome with nanomolar
sified as caspase-like, trypsin-like, and chymotrypsin-like,
potency23 although synthetic analogs show only moderate
respectively) catalyze proteolysis of substrates by nucleophilic
potency.24 Several dipeptide inhibitors25–32 have been described
attack of the hydroxyl group of the N-terminal threonine resi-
(Fig. 2), notably trimethoxy-L-phenylalanine derivative (1) which
dues (Thr1Og) on peptide bonds.6,7 Numerous 20S inhibitors
have been described8 that form covalent adducts with the active
has been shown to inhibit the b5 activity reversibly.26–29 Recently,
we described the biological properties of screening hit 2,32
site threonine hydroxyls including peptidic aldehydes, vinyl
sulfones,3 vinylacylpyrroles,9 a,b-epoxyketones10,11 such as car-
filzomib (Fig. 1), 2-keto-1,3,4-oxadiazoles,12 b-lactones13–15 e.g.
a rapid-equilibrium inhibitor of the b5 (chymotrypsin-like)
activity of the proteasome that does not inhibit the activity of the
b1 or b2 sites. Compound 2 also inhibits TNFa-induced acti-
salinosporamide A, b-lactams16 and boronic acids.17–21 The
vation of NFkB-Luc in HEK293 cells consistent with protea-
dipeptide boronic acid bortezomib is used clinically to treat
some inhibition and is cytotoxic toward human cancer cell lines.
multiple myeloma and relapsed mantle cell lymphoma, and is
being evaluated for the treatment of other malignancies.22 Bor-
We therefore investigated dipeptide analogs of 2 preparing over
1500 derivatives using automated solution-phase synthesis33 and
tezomib forms a slowly reversible adduct with the N-terminal
reported the detailed biological properties of fifteen compounds
threonine of the 20S b5 site (Ki ¼ 0.55 nM; dissociation t1/2 ca.
representative of several sub-series32 followed by a description of
the SAR of the P3 Thr series including potent non-covalent
inhibitors such as 3 and 4.34 Crystal structures of several inhib-
itors in complex with yeast 20S demonstrated the non-covalent
binding interactions with the b5 sub-unit and suggested that
replacement of the P3 Thr by a bulky hydrophobic residue might
lead to a more optimal occupancy of the S3 binding pocket. Of
several residues investigated at this position,35 the b-neopentyl
asparagine group, previously incorporated only in covalent
110 min)7,8 which has been characterized by X-ray
Discovery, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street,
Cambridge, MA 02139, USA. E-mail: blackburn@mpi.com; Tel: +1 617
761 6811
† Electronic
supplementary
information
(ESI)
available:
Crystallographic data and refinement statistics. See DOI:
10.1039/c2md20060k
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Fig. 1 Covalent proteasome inhibitors.
inhibitors12 leads to potent, selective and reversible inhibition of
hydrogenolysis, and the resulting acid 39 coupled to tert-butyl-
hydroxyamidine and cyclized36 by microwave irradiation in
pyridine solution to give 40 which was further elaborated to
compounds 36 and 37. Coupling the side-chain acid of 39 to n-
propylamine or isobutylamine, deprotection of the N-terminal
amino group and aroylation afforded 34 and 35, respectively.
the b5 sub-unit of 20S as discussed in this paper.
2. Chemistry
Initially, we approached the further optimization of dipeptides
by retaining the P1 benzylic and P2 homoPhe residues previously
shown to contribute to the potency of compounds such as 3 and 4
while replacing the P3 Thr with larger residues. The key amino
acid amides 5 were prepared as shown in Scheme 1. Coupling of
5a with an orthogonally protected diaminopropanoic acid fol-
lowed by removal of the side-chain CBZ protecting group gave
6b which was treated with dimethylbutanoic acid to install the
branched P3 residue. Deprotection of the terminal amino group
followed by N-terminal capping with three aryl carboxylic acids
afforded analogs 9–11.
3. Results and discussion
The inhibitory activities of compounds 9–37 on the rate of
hydrolysis of the b5 substrate Ac-WLA-AMC catalyzed by
human 20S constitutive proteasome were determined and selec-
tivity assessed by measuring inhibition of the hydrolysis of b1
(Z-LLE-AMC) and b2 (Ac-KQL-AMC) selective substrates.32
Potencies were also determined in the Proteasome-Gloꢀ cell-
based assay that measures directly the peptidase activity of the b5
site of the 26S proteasome in cells using a luminogenic chymo-
trypsin-like substrate. We also assessed the effects of the
compounds on activation of the transcription factor NFkB30,37 in
cells in conjunction with cell viability experiments in which
cellular ATP levels were determined by ATPlite assay in the
human lung cancer cell line Calu6 (Table 1). In our earlier work
on related dipeptides32–35 we found that compounds with rela-
tively small P3 residues exhibit little 20S inhibition unless
a compensating large aryl-containing P4 residue is present. For
The key intermediate towards P3 neopentyl asparagine
derivatives, N-a-Boc-b-neopentyl asparagine 8 was prepared as
described previously32 and incorporated into peptides by solu-
tion phase parallel synthesis as shown in Scheme 2.
Analogs 34 and 35 in which the neopentyl amide side-chain
was replaced by smaller alkyl groups and heterocyclic replace-
ments for the neopentyl amide side-chain (oxadiazoles 36 and 37)
were also investigated (Scheme 3). Thus, the side-chain protected
aspartic acid derivative 38 was prepared, subjected to
Fig. 2 Non-covalent proteasome inhibitors.
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Scheme 1 Synthesis of P3-diaminopropanoic acid peptides. Reagents and conditions: (i) R1NH2, HBTU, NMM, DCM; (ii) 4 M HCl in dioxane; (iii) 5a,
HATU, NMM, DCM, 25 ꢀC, 16 h; (iv) H2 (1 atm), 10% Pd–C, i-PrOH, 4 h; (v) Me3CH2COOH, HATU, NMM, DCM, 25 ꢀC, 16 h; (vi) RCOOH,
HBTU, NMM, DCM.
example, while compounds 3 and 4 possess respectable potencies,
analogous compounds with simple acyl groups at the P4 position
invariably show IC50 values >1 mM. In contrast, all the P3 neo-
pentyl Asn derivatives, including analogs 12 and 13 with small P4
residues, show relatively high b5 inhibitory potencies without
any effect on b1 or b2 activity (Table 1). When the P4 residue was
extended with alicyclic (compound 14) or aryl residues (analogs
15–20) significant improvements in inhibitory activity were
observed. In general, we observed a good correlation between the
inhibition of 20S b5 activity in vitro and proteasome inhibition in
cells as measured by the Proteasome-Glo and NFkB-Luc assays.
Derivatives 18 and 19 are considerably more potent inhibitors
than their P3 Thr counterparts (compounds 3 and 4 respectively)
in all four assays, particularly in cells. The reverse amide analogs,
namely diaminopropionic acid derivatives 9–11, proved to be
considerably less active and this series was not pursued further.
Interestingly, while the large hydrophobic P4 residues improved
potency of the neopentyl Asn series, they were not essential as
demonstrated by the inhibitory activities of isoxazole derivative
20. Reduced inhibitory activity resulted from replacement of the
P1 benzylic group by the smaller cyclopropylmethyl (compare
compounds 16 and 21). The importance of the branched P3 side-
chain for enzyme inhibition was demonstrated by the consider-
ably lower activity of the n-propyl compound 34 while the
iso-butyl derivative 35 had comparable enzyme inhibitory
activity to neopentyl analog 20. Attempts to find potent
heterocyclic replacements for the neopentyl Asn side-chain were
largely unsuccessful35 with the exception of the tert-butyl-1,2,4-
oxadiazole isostere. Even here, compounds 36 and 37 showed
significant reductions in activity in comparison with their neo-
pentyl Asn analogs 19 and 20.
A co-crystal of compound 20, in complex with yeast open gate
20S is depicted in Fig. 3. We observed the same general non-
covalent binding mode that we reported earlier for compound
Scheme 2 Synthesis of P3-neopentyl asparagine peptides. Reagents and conditions: (i) neopentylamine, HOBt, NMM, DCM, 25 ꢀC, 24 h; (ii) H2 (1 atm),
10% Pd/C, MeOH, 25 ꢀC, 3 h; (iii) 5a–g, HBTU, NMM, DMF, 25 ꢀC, 24 h; (iv) 4 N HCl, dioxane, 2 h; (v) RCOOH, HBTU, NMM, DMF, 25 ꢀC, 24 h.
712 | Med. Chem. Commun., 2012, 3, 710–719
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Scheme 3 Synthesis of P3 asparagine and oxadiazole-containing peptides. Reagents and conditions: (i) 5a, HBTU, NMM, DMF, 25 ꢀC, 16 h. (ii) H2,
10% Pd–C, MeOH, 1 atm; (iii) t-Bu(NH2)C]NOH, EDC, DCM, DMF, 25 ꢀC, 24 h; (iv) pyridine, 110 ꢀC, MWI, 20 min; (v) 4 N HCl, dioxane, 2 h; (vi)
RCOOH, HBTU, NMM, DMF; (vii) RNH2, HBTU, NMM, DMF.
2932–34 including backbone H-bonding interactions with several
residues of the b5 sub-unit, viz. Gly47, Thr21 and (via a bridging
water molecule) Ala49 and Ala50. There is also evidence for an
interaction between the carbonyl oxygen of the neopentyl Asn
side-chain and Ser118 of the b6 sub-unit (Fig. 3a). The P1 benzyl
group occupies a well-defined S1 binding pocket with an addi-
tional hydrophobic interaction taking place between the 40-
methyl group and a sub-pocket of S1. Although P2 residues point
out toward solvent, the two methylene groups of homoPhe
permit a conformation in which the p-system of the aryl group
forms a ledge interaction with Gly47-Gly48. The very large S4
pocket can be partially occupied by a range of N-terminal resi-
dues that likely contribute to potency by picking up further
hydrophobic interactions. We attribute the generally high
potencies of compounds in this series to the near optimal fit of
the neopentyl-Asn P3 substituent in the S3 binding pocket of the
b5 sub-unit (Fig. 3b). A crystal structure of yeast 20S in complex
with tert-butyloxadiazole 37 was also obtained and the binding
mode is compared with that of P3 neopentyl Asn analog 20 in
Fig. 4. While the backbone conformations of these compounds in
the bound mode are very similar, the pendant tert-butyl group of
37 displaces His98 of the b6 sub-unit rather than projecting
optimally into the b5 S3 binding pocket accounting for the loss in
inhibitory activity.
We next investigated analogs with an 20-chlorobenzyl residue
in the P1 position reasoning that the interaction of chlorine with
a S1 sub-pocket observed in earlier series32,34 would contribute to
affinity and that these derivatives would be less susceptible to
oxidative metabolism than the 40-methyl counterpart. (Incuba-
tion of compound 18, for example, with human or rodent liver
microsomes followed by LC-MS-MS analysis indicated that ring
hydroxylation of the P1 and P2 residues contributes to low
Fig. 3 (a) Schematic representation of the b5/b6 active site of 20S with compound 20 bound. Key hydrogen bonds between the inhibitor and the protein
are shown as dashed lines colored magenta, with distances indicated in Angstroms. (b) Molecular surface of 20S with compound 20 bound, showing
specificity pockets S1–S4. Atoms are shown in ball and stick representation with carbon in yellow, nitrogen in blue and oxygen in red. The side chain
atoms for Thr2 (b5) and Asp114 (b6) are shown with carbon colored green. Figure made using PyMOL Molecular Graphics System (DeLano Scientific,
Palo Alto, CA).
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Table 1 Enzymatic, cellular and anti-proliferative effects of P3 neopentyl Asn and Thr dipeptides compared with boronates
Human 20S
Calu6 b5c
NFkB-Luc Calu6
Human 20S
Calu6
NFkB-Luc Calu6
Compound
b5c IC50 (nM)a IC50 (nM)b IC50 (nM)c LC50 (nM)d Compound b5c IC50 (nM)a b5c IC50 (nM)b IC50 (nM)c LC50 (nM)d
Bortezomib8 2.0
3.9
9.0
53.0
159
14
ND
ND
ND
960
610
30
73
17
40
11
9.7
25
9.7 (100)
6.2 (100)
45 (79)
120 (81)
90 (100)
ND
5.2
21
22
23
24
25
26
12
4.0
3.0
11
3.7
2.1
4.0
3.9
1.2
33.3
22.6
16.0
23.0
360
85
18
12
11
13
35
24
9.5
940
690
ND
1400
ND
ND
30
38 (86)
110 (80)
18 (86)
39 (90)
17 (98)
9 (85)
150
94
20
4.7
2.4
41
39
61
MLN2238
3.0
14d
130
280
200
ND
ND
ND
890
210
180
120
89
232
334
434
9
16.0
20.0
7.3
>1000
>1000
910
50
10
11
12
13
14
15
16
17
1832
19
20
ND
ND
2732
2832
2932
30
31
32
33
34
10 (74)
9 (77)
150 (80)
62 (90)
62 (85)
23 (79)
12 (80)
32 (86)
22 (78)
25 (97)
9 (85)
12.0 (92)
950 (85)
140 (80)
320 (93)
120 (80)
ND
53 (88)
110 (98)
900 (82)
6
25
12
8.4
5.4
210
170
1100
200
ND
250
36
22
6.8
4.8
4.7
31
10.0
2.6
160
35
36
37
7.3
11.6
93
27
1700
1500
a
Human erythrocyte 20S was used as a source of constitutive proteasome. All compounds showed negligible inhibition (IC50 values >10 000 nM) of the
cleavage of b1 (Z-LLE-AMC) and b2 (Ac-KQL-AMC) substrates. Data are means of at least three independent measurements. Cell-based
b
c
(Proteasome-Glo) assay measuring peptidase activity of the b5 site of the 26S proteasome. Inhibition of NFkB-luciferase activity in HEK293 cells
d
stimulated with TNF-a; % maximal inhibition values are shown in parentheses.32 Anti-proliferative effects in Calu6 ATPlite (Perkin Elmer) cell
viability assay. Data for compound 4 acquired with H460 cells. ND not determined.
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4. Conclusions
In summary, we have found that dipeptides with a P3 neopentyl
Asn residue are reversible non-covalent inhibitors of the b5 sub-
unit of 20S with unprecedented potency and selectivity over the
human b1 and b2 sub-units (IC50 > 10 mM) attributable to the
near optimal fit of this substituent in the S3 binding pocket of the
20S b5 sub-unit. In general, we observed a good correlation
between the inhibitory IC50 values for 20S b5 activity in vitro and
proteasome inhibition in cells as measured by the Proteasome-
Gloꢀ assay and by NFkB-Luc activity with several derivatives
showing improved cellular activities compared to tripeptide 8
and some of the compounds (for example, 18, 19, 25 and 29)
having comparable potencies to the boronates. The cytotoxic
LC50 values of these compounds in Calu6 cells also correlate with
their 20S b5 potencies and support the premise that selective non-
covalent inhibition of the b5 (chymotrypsin-like) site of the
proteasome is sufficient to inhibit TNFa-dependent NFkB
activity and the proliferation of cancer cells.
Fig. 4 Superposition of the structures for compounds 20 and 37 bound
to the b5/b6 active site of 20S, highlighting the S3 pocket and changes in
the position of His98. All residues labeled are from the b6 sub-unit, with
the exception of Val31 (b5). Coloring scheme is as described above, with
carbon atoms for 20 shown in yellow and 37 in green.
5. Experimental
Compound synthesis and characterization
General. NMR spectra were recorded on a Bruker 300 MHz
Avance1 or 400 MHz Avance2 (5 mm QnProbe) using residual
solvent peaks as the reference. Compound purity was determined
by analysis of the diode array UV trace of an LC-MS spectrum
using the following procedure. Compounds were dissolved in
DMSO, methanol or acetonitrile and the solutions were analyzed
using an Agilent 1100 LC interfaced to a micromass Watersꢁ
Micromassꢁ Zsprayꢀ Mass Detector (ZMD). One of two
gradients was used to elute the compounds either a formic acid
(FA) gradient (acetonitrile containing 0 to 100 percent 0.1%
formic acid in water) or an ammonium acetate (AA) gradient
(acetonitrile containing zero to 100 percent 10 mM ammonium
acetate in water). All compounds were determined to be >95%
pure. High-resolution mass spectra were recorded using
a QSTARꢁ XL quadrupole time-of-flight mass spectrometer
(Applied Biosystems) coupled to an 1100 series HPLC system
(Agilent Technologies). An isocratic flow of 40% H2O 60%
MeCN containing 0.1% HOAc at 200 mL minꢁ1 was used to
deliver each sample to the electrospray source tuned in ESI TOF-
MS positive mode with a 3 min acquisition time for each analysis.
The mass spectrometer was externally calibrated between m/z
400–800 by using API Calibration (NaCsI) Solution. For each
analysis, approximately 5 scans were summed and the centroid
m/z value of the protonated monoisotopic molecular ion [M +
H]+ was recorded. For each compound, the experimental and
calculated [M + H] values were within ꢂ4 ppm, supporting their
expected elemental composition.
metabolic stability (extraction ratio Eh > 0.9) of this and related
compounds.) In general, the same trends in inhibitory activity as
a function of P4 residue were observed as with the previous
series. Thus, with homoPhe as the P2 residue, the pairs of
compounds 15 and 22; 16 and 23; 18 and 24; 19 and 25; 20 and 26
showed comparable potencies in each of the four assays. We also
investigated P2 residues with a lower potential for oxidative
metabolism by replacing homoPhe by Ala or by electron defi-
cient pyridyl residues. In the case of P4 phenylpropionyl deriv-
atives 27 and 28 loss of the ledge interaction present with 23 had
little effect on enzymatic or cellular potency whereas in the iso-
xazole series analogs 30, 31, 32 and 33 were less potent than the
homoPhe analog 26. In contrast, P2 4-pyridylalanine derivative
29 was found to have comparable potencies to homoPhe analog
25. Indeed, compounds 25 and 29 showed the best combination
of enzymatic and cellular activities of all the compounds
prepared to date including anti-proliferative effects comparable
to those of the boronates. The reason that P4 ketoindole residues
confer high potencies is unclear though we have shown previ-
ously32 that there is no evidence of any covalent interactions with
20S and thus the group presumably acts as a constrained version
of the phenylpropionyl residue. As well as demonstrating selec-
tive inhibition of the chymotryptic activity of the proteasome,
this series of compounds is selective for unrelated proteases. For
example, compounds 25 and 26 did not show any significant
inhibitory activity on a panel of purified human proteases
including cathepsin B and L, coagulation factor b-XIIa,
chymotrypsin, elastase, plasmin, thrombin, tissue plasminogen
activator, trypsin and calpain I at concentrations of 100 mM.
Unfortunately, although compounds such as 29 designed to be
metabolically more stable afforded fewer metabolites in micro-
somal stability experiments extraction ratios remained very high
due to oxidative metabolism of the neopentyl group.
General synthetic procedures
(S)-2-(tert-Butoxycarbonylamino)-4-(neopentylamino)-4-oxo-
butanoic acid (8) and C-terminal capped amino acids (5) were
prepared as described previously.32
(S)-2-Amino-N-(4-methylbenzyl)-4-phenylbutanamide hydro-
ꢀ
chloride (5a): mp 109–112 C. Found C, 67.28%; H, 6.74%; N,
8.65%: calcd for C18H23ClN2O C, 67.81%; H, 7.27%; N, 8.79%.
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1
(S)-2-Amino-N-(2-chlorobenzyl)-4-phenylbutanamide hydro-
ꢀ
9: HMR (400 MHz, CD3OD) d: 1.05 (s, 9H), 1.98 (m, 1H),
2.13 (s, 2H), 2.29 (s, 3H), 2.47 (t, J ¼ 8 Hz, 2H), 2.66 (m, 2H),
2.87 (t, J ¼ 8 Hz, 2H), 3.44 (m, 1H), 3.62 (m, 1H), 4.36 (m, 2H),
4.49 (t, 1H, J ¼ 8 Hz), 7.10–7.28 (m, 9H).
chloride (5b): mp 166–167 C. Found C, 60.14%; H, 5.80%; N,
8.24%: calcd for C17H20Cl2N2O; C, 60.18%; H, 5.94%; N, 8.26%.
1
11: HMR (400 MHz, d8-THF) d: 9.00 (s, 1H), 8.39 (m, 1H),
Preparation of benzyl tert-butyl ((S)-3-(((S)-1-((4-methylbenzyl)
amino)-1-oxo-4-phenylbutan-2-yl)amino)-3-oxopropane-1,2-
diyl)dicarbamate (6a)
7.43 (m, 1H), 7.23–7.04 (m, 11H), 4.51–4.25 (m, 4H), 3.91 (m,
1H), 3.53 (m, 1H), 2.64 (m, 1H), 2.00 (m, 4H), 1.85 (m, 2H), 1.00
(s, 9H).
A solution of the dicyclohexylamine salt of N-a-Boc-CBZ-dia-
minopropionic acid (1.0 g, 1.9 mmol) in DMF (7 mL) and
dichloromethane (3 mL) was treated with 5a hydrochloride
(0.58 g, 1.83 mmol), HATU (0.695 g, 1.83 mmol), N-methyl-
morpholine (0.6 mL, 5.5 mmol) and stirred at ambient tempera-
ture for 16 h. The DCM was evaporated under reduced pressure
and the residual solution poured onto ice cold saturated NaHCO3
solution and the precipitate collected and recrystallized from ethyl
acetate to give 6a (0.65 g, 60%) 1HMR (400 MHz, d6-DMSO) d:
8.30 (m, 1H), 8.10 (m, 1H), 7.37–7.06 (m, 15H), 6.95 (m, 1H), 4.95
(m, 2H), 4.24 (m, 2H), 4.09 (m, 1H), 2.55 (m, 2H), 2.25 (s, 3H), 1.98
(m, 1H), 1.85 (m, 1H), 1.38 (s, 9H).
Synthesis of capped peptides 9–35
Compounds were prepared using the solution phase parallel
amide synthesis techniques described previously.32,34 Amide
couplings were effected using HBTU and N-methylmorpholine
(NMM) in DMF solution. After evaporation of the solvent in
a
Genevac HT-12, crude products were dissolved in
CHCl3 : THF (3 : 1) and extracted with saturated aqueous
NaHCO3. All final compounds were purified by preparative
reverse phase HPLC using mass-directed fraction collection
conducted on an Agilent 1100 series LC/MSD instrument using
a Waters SunFire C-18 5 mm preparative OBD column (19 ꢃ
150 mm). The compounds were eluted with a water–MeCN
gradient (0.1% formic acid) optimized by the A2Prep Agilent
software. Final products described in the manuscript were
Preparation of compounds 9–11
Compound 6a (181 mg, 0.3 mmol) was dissolved in isopropanol
(50 mL), with heating, treated with 10% Pd/carbon and hydro-
genated at balloon pressure for 16 h. The catalyst was removed
and the solvent evaporated under reduced pressure to give 6b
LC-MS [M + H] 470 which was redissolved in DMF (3.0 mL)
and treated with tert-butylacetic acid (42 mL, 0.33 mmol), HATU
(125 mg, 0.33 mmol) and diisopropylethylamine (108 mL, 0.66
mmol) and stirred at ambient temperature for 16 h. The reaction
mixture was diluted with ice cold saturated aqueous sodium
bicarbonate solution and the resulting precipitate collected. The
crude product was purified by reverse phase chromatography on
a C-18 column eluting with water–MeCN gradient (0.1% formic
acid) and the desired fractions lyophilized to give 7 (144 mg,
85%). 1H NMR (400 MHz, d4-MeOH) d: 7.3–7.10 (m, 9H), 4.40–
4.30 (m, 2H), 4.20 (m, 1H), 3.50–3.45 (m, 2H), 3.34–3.31 (m, 4H),
2.74–2.60 (m, 1H), 2.31 (s, 2H), 2.15–1.94 (m, 4H), 1.44 (s, 9H),
1.01 (s, 9H).
1
further characterized by H NMR spectroscopy and high reso-
lution mass spectrometry.
1
32: HMR (400 MHz, CD3OD) d: 0.83 (s, 9H), 2.51 (s, 3H),
2.73 (dd, J ¼ 12 Hz, J ¼ 7 Hz, 1H), 2.86 (dd, J ¼ 12 Hz, J ¼ 7 Hz,
1H), 2.90 (s, 2H), 2.95 (dd, J ¼ 12 Hz, J ¼ 7 Hz, 1H), 3.22 (dd,
J ¼ 12 Hz, J ¼ 7 Hz, 1H), 4.48 (m, 2H), 4.66 (m, 1H), 6.46
(s, 1H), 7.15–7.25 (m, 7H), 7.39 (m, 1H).
In some cases compounds prepared by parallel synthesis as
described above were scaled-up using conventional techniques as
described below.
(S)-N1-((S)-1-(4-Methylbenzylamino)-1-oxo-4-phenylbutan-2-yl)-
2-(5-methylisoxazole-3-carboxamido)-N4-neopentylsuccinamide
(20)
A solution of 7 (31 mg, 0.056 mmol) in dioxane (1.0 mL) was
treated with 4 M HCl in dioxane (1.0 mL) and stirred at ambient
temperature for 2 h whereupon LC-MS analysis ([M + H] 467)
indicated that deprotection was complete. The reaction mixture
was evaporated under reduced pressure and the residue
suspended in dichloromethane (3 mL) and treated with N-
methylmorpholine (18 mL, 0.157 mmol), 5-methylisoxazole-3-
carboxylic acid (7.8 mg, 0.061 mmol) and a solution of HBTU
(23 mg, 0.061 mmol) in DMF (0.5 mL). After stirring for 16 h at
ambient temperature, the dichloromethane was evaporated and
the residue diluted with ice cold saturated aqueous sodium
bicarbonate solution and the resulting precipitate collected. The
crude product was purified by reverse phase chromatography on
a C-18 column eluting with water–MeCN gradient (0.1% formic
acid) and the desired fractions lyophilized to give 10 (27 mg,
87%). LC-MS [M + H] 576. 1H NMR (400 MHz, d4-MeOH) 7.3–
7.0 (m, 9H), 6.34 (s, 1H), 4.56 (t, 1H), 4.27 (m, 1H), 4.25 (s, 2H),
3.58 (m, 2H), 2.56 (m, 2H), 2.38 (s, 3H), 2.20 (s, 3H), 2.10–1.82
(m, 4H), 0.86 (s, 9H).
A suspension of 5-methylisoxazole-3-carboxylic acid (0.017 g,
0.13 mmol) in anhydrous dichloromethane (1.0 mL) was
treated with DMF (1.8 mL, 0.024 mmol) followed by oxalyl
chloride (12 mL, 0.14 mmol) and stirred at ambient temperature
for 1.5 h. Volatiles were removed under vacuo and the residue
re-dissolved in dichloromethane (1 mL) and added to a solution
of (2R)-2-amino-Nꢄ4ꢄ-(2,2-dimethylpropyl)-Nꢄ1ꢄ-{(2R)-1-
[(4-methylbenzyl)amino]-1-oxo-4-phenylbutan-2-yl}succinamide
(0.06 g, 0.1 mmol) in dichloromethane (1.0 mL). N-Methyl-
morpholine (0.1 mL, 0.9 mmol) was next added and the
mixture was stirred at ambient temperature for 4 h. The
resulting suspension was diluted with dichloromethane–hexane
1 : 1 (3 mL) and the precipitate collected. The crude product
was dissolved in DCM–MeOH applied to silica gel thick layer
plates and eluted with 20% ethyl acetate in dichloromethane.
Extraction of the main band afforded 20 (48 mg, 83%). 1H
NMR (400 MHz, CD3OD) d: 0.83 (s, 9H), 1.96 (m, 2H), 2.20
(m, 2H), 2.28 (s, 3H), 2.46 (s, 3H), 2.62 (m, 1H), 2.68 (m, 1H),
2.81 (m, 1H), 2.88 (s, 3H), 2.92 (m, 1H), 4.33 (m, 3H), 4.90 (m,
Compounds 9 and 11 were prepared similarly.
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2H), 6.45 (s, 1H), 7.15 (m, 9H). HRMS (ESI+): calculated for
C32H41N5O5 [M + H] 576.3180; found 576.3185.
dioxane (2.0 mL) and the mixture allowed to warm to ambient
temperature. After stirring for 2 h, the solvent was evaporated
and the residue treated with a solution of 5-methylisoxazole-3-
carboxylic acid (0.036 g, 0.28 mmol), HBTU (0.148 g, 0.39 mmol)
and N-methylmorpholine (57 mL, 0.5 mmol) in DMF (2.0 mL).
The reaction mixture was stirred at ambient temperature for 4 h
then diluted with water (100 mL) and extracted with dichloro-
methane. The extracts were dried (MgSO4) and evaporated and
the resulting crude product purified by reverse phase HPLC a on
a C-18 column eluting with water–MeCN gradient to give N-((S)-
3-(3-tert-butyl-1,2,4-oxadiazol-5-yl)-1-((S)-1-(4-methylbenzylamino)-
1-oxo-4-phenylbutan-2-ylamino)-1-oxopropan-2-yl)-5-methylis-
oxazole-3-carboxamide (37) (70 mg, 46%). 1H NMR (300 MHz,
CDCl3) d: 1.32 (s, 9H), 1.95 (m, 1H), 2.15 (m, 1H), 2.31 (s, 3H),
2.47 (s, 3H), 2.58 (m, 2H), 3.15 (m, 1H), 3.50 (m, 1H), 4.34 (m,
3H), 5.08 (m, 1H), 6.24 (m, 1H), 6.40 (s, 1H), 7.10 (m, 5H), 7.21
(m, 5H), 8.25 (m, 1H). HRMS (ESI+): calculated for
C32H38N6O5 [M + H] 587.2976; found 587.2982.
34: 1HMR (400 MHz, CD3OD) d: 0.75 (t, J ¼ 8 Hz, 3H), 1.33
(m, 2H), 1.85 (m, 2H), 2.10 (m, 2H), 2.21 (s, 3H), 2.39 (s, 3H), 2.53
(m, 1H), 2.63 (m, 1H), 2.76 (m, 1H), 2.92 (t, J ¼ 9 Hz, 2H), 4.21
(m, 1H), 4.24 (s, 2H), 4.80 (m, 1H), 6.37 (s, 3H), 7.06 (m, 9H).
35: 1HMR (400 MHz, CD3OD) d: 0.74 (d, J ¼ 8 Hz, 6H), 1.58
(m, 1H), 1.85 (m, 2H), 2.10 (m, 2H), 2.20 (s, 3H), 2.38 (s, 3H),
2.70–2.48 (m, 3H), 2.78 (m, 2H), 4.21 (m, 1H), 4.24 (s, 2H), 4.80
(m, 1H), 6.37 (s, 3H), 7.06 (m, 9H).
Benzyl(3S)-3-[(tert-butoxycarbonyl)amino]-4-[((1S)-1-{[(4-
methylbenzyl)amino]carbonyl}-3-phenylpropyl)amino]-4-
oxobutanoate (38)
2-Amino-N-(4-methylbenzyl)-4-phenylbutanamide hydrochlo-
ride (5a)32 (1.5 g, 4.7 mmol) and N-(tert-butoxycarbonyl)-L-
aspartic acid 4-benzyl ester (1.60 g, 4.94 mmol) in N,N-dime-
thylformamide (5.0 mL) was treated with HBTU (1.82 g, 4.80
mmol) and N-methylmorpholine (1.55 mL, 4.1 mmol) and stirred
at ambient temperature for 16 h. The reaction mixture was
poured into cold saturated NaHCO3 solution and the precipitate
collected, dried over CaSO4 and subjected to chromatography on
silica gel eluting with hexane–EtOAc gradient to give benzyl (3S)-
3-[(tert-butoxycarbonyl)amino]-4-[((1S)-1-{[(4-methylbenzyl)amino]-
carbonyl}-3-phenylpropyl)amino]-4-oxobutanoate 38 (2.5 g,
1
91%). HMR (400 MHz, CDCl3) d: 1.44 (s, 9H), 1.98 (m, 1H),
2.28 (s, 3H), 2.65 (m, 2H), 2.75 (m, 1H), 2.87 (m, 1H), 4.35 (m,
1H), 4.37 (m, 1H), 4.47 (m, 2H), 5.00 (m, 2H), 5.52 (m, 1H), 6.86
(m, 1H), 7.00 (d, 1H), 7.12 (m, 8H), 7.30 (m, 7H).
1
32: HMR (400 MHz, CD3OD) d: 0.83 (s, 9H), 2.51 (s, 3H),
2.73 (dd, J ¼ 12 Hz, J ¼ 7 Hz, 1H), 2.86 (dd, J ¼ 12 Hz, J ¼ 7 Hz,
1H), 2.90 (s, 2H) 2.95 (dd, J ¼ 12 Hz, J ¼ 7 Hz, 1H), 3.22 (dd, J ¼
12 Hz, J ¼ 7 Hz, 1H), 4.48 (m, 2H), 4.66 (m, 1H), 6.46 (s, 1H),
7.15–7.25 (m, 7H), 7.39 (m, 1H).
High Resolution Mass Spectrometry (HRMS) data
tert-Butyl (S)-3-(3-tert-butyl-1,2,4-oxadiazol-5-yl)-1-((S)-1-(4-
methylbenzylamino)-1-oxo-4-phenylbutan-2-ylamino)-1-
oxopropan-2-ylcarbamate (40)
Compound 9: HRMS (ESI+): calculated for C36H46N4O4
[M + H] 599.3597; found 599.3585.
Compound 12: HRMS (ESI+): calculated for C29H40N4O4
[M + H] 509.3122; found 509.3129.
A solution of 38 (0.400 g, 0.681 mmol) in ethanol (50 mL) was
treated with 10% palladium on carbon and subjected to hydro-
genation under balloon pressure. After stirring at ambient
temperature for 3 h the catalyst was removed by filtration and the
solvent evaporated to give (3S)-3-[(tert-butoxycarbonyl)amino]-
4-[((1S)-1-{[(4-methylbenzyl)amino]carbonyl}-3-phenylpropyl)
amino]-4-oxobutanoic acid (39) (0.334 g, 98%) which was used
without further purification. To a solution of compound 39
(0.225 g, 0.452 mmol) in dichloromethane (3.0 mL) and DMF
(1.0 mL) was added N0-hydroxy-2,2-dimethylpropanimidamide
(0.0625 g, 0.54 mmol) followed by N-(3-dimethylaminopropyl)-
N0-ethylcarbodiimide hydrochloride (0.103 g, 0.54 mmol). The
mixture was stirred for 16 hours and the dichloromethane
evaporated under vacuo. The residue was treated with pyridine
(3 mL) and heated under microwave irradiation at 110 ꢀC for
20 min. The volatiles were evaporated and the residue subjected
to preparative reverse phase HPLC on a C-18 column eluting
with water–MeCN gradient to give 40 (150 mg, 57%). 1H NMR
(400 MHz, d4-MeOH) d: 1.32 (s, 9H), 1.41 (s, 9H), 1.97 (m, 1H),
2.10 (m, 1H), 2.29 (s, 3H), 2.62 (m, 2H), 3.30 (m, 2H), 4.33 (m,
3H), 4.63 (m, 1H), 7.13 (m, 7H), 7.22 (m, 2H).
Compound 13: HRMS (ESI+): calculated for C32H46N4O4
[M + H] 551.3592; found 551.3596.
Compound 15: HRMS (ESI+): calculated for C35H43FN4O4
[M + H] 603.3341; found 603.3347.
Compound 16: HRMS (ESI+): calculated for C36H46N4O4
[M + H] 599.3592; found 599.3597.
Compound 17: HRMS (ESI+): calculated for C38H50N4O4
[M + H] 627.3905; found 627.3908.
Compound 19: HRMS (ESI+): calculated for C37H43N5O5
[M + H] 638.3337; found 638.3343.
Compound 20: HRMS (ESI+): calculated for C32H41N5O5
[M + H] 576.3180; found 576.3185.
Compound 21: HRMS (ESI+): calculated for C32H44N4O4
[M + H] 549.3435; found 549.3430.
Compound 22: HRMS (ESI+): calculated for C34H40ClFN4O4
[M + H] 623.2795; found 623.2799.
Compound 23: HRMS (ESI+): calculated for C35H43ClN4O4
[M + H] 619.3046; found 619.3054.
Compound 24: HRMS (ESI+): calculated for C40H45ClN4O5
[M + H] 697.3151; found 697.3157.
Compound 25: HRMS (ESI+): calculated for C36H40ClN5O5
[M + H] 658.2791; found 658.2799.
N-((S)-3-(3-tert-Butyl-1,2,4-oxadiazol-5-yl)-1-((S)-1-(4-methyl-
benzylamino)-1-oxo-4-phenylbutan-2-ylamino)-1-oxopropan-2-
yl)-5-methylisoxazole-3-carboxamide (37)
Compound 26: HRMS (ESI+): calculated for C31H38ClN5O5
[M + H] 596.2640; found 596.2648.
A solution of 40 (0.15 g, 0.26 mmol) in 1,4-dioxane (1 mL) was
Compound 30: HRMS (ESI+): calculated for C29H35ClN6O5
[M + H]; 583.2430; found 583.2432.
cooled in an ice bath and treated with a solution of 4 M HCl in
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Compound 31: HRMS (ESI+): calculated for C29H35ClN6O5
[M + H] 583.2430; found 583.2434.
following selective substrates were obtained from AnaSpec, Inc.
with the exception of Z-LLE-AMC which was from Boston
Biochemꢀ and were each used at a final concentration of 15 mM
to assay the constitutive (c) and sub-sites: b1c, Z-LLE-AMC;
b2c, Ac-KQL-AMC; b5c, Ac-WLA-AMC. Reactions were per-
formed at 37 ꢀC in 384-well black microtiter plates (Corning Inc.)
using 0.25 nM 20S and 12 nM PA28a in a final volume of 50 mL
buffer containing 20 mM HEPES, pH 7.4, 0.5 mM EDTA and
0.01% BSA. Peptidase activity was measured by monitoring
AMC liberation over time with a Polarstar Galaxy fluorimeter
(BMG Labtechnologies) using excitation and emission wave-
lengths of 340 nm and 460 nm, respectively. Percentage inhibi-
tion of 20S activity was calculated relative to the controls DMSO
and 10 mM ML599698, an analog of bortezomib that contains
a phenyl group in place of the N-terminal pyrazine cap.
Compound 32: HRMS (ESI+): calculated for C30H35Cl2N5O5
[M + H] 616.2093; found 616.2090.
Compound 33: HRMS (ESI+): calculated for C24H32ClN5O5
[M + H] 506.2165: found 506.2169.
Compound 34: HRMS (ESI+): calculated for C30H37N5O5
[M + H] 584.2873: found 584.2867.
Compound 35: HRMS (ESI+): calculated for C31H39N5O5
[M + H] 562.3029: found 562.3026.
Compound 35: HRMS (ESI+): calculated for C31H39N5O5
[M + H] 562.3029; found 562.3026.
Compound 36: HRMS (ESI+): calculated for C37H40N6O5
[M + H] 649.3133; found 649.3139.
Compound 37: HRMS (ESI+): calculated for C32H38N6O5
[M + H] 587.2976; found 587.2982.
We have reported the characterization of compounds 14, 18,
27, 28, 29 previously.32
X-ray crystallography. Crystals of purified wild-type and open-
gated mutant 20S proteasome were grown in hanging drops at
room temp, as described previously,32 using a drop volume of
1.5 mL 20S (20 mg mLꢁ1 in 10 mM Tris–HCl, pH 7.5, and 1 mM
EDTA) and 0.5 mL of reservoir solution containing 100 mM
MES, pH 7.0, 40 mM Mg(CH3COO)2, 15% 2-methyl-2,4-pen-
tanediol (MPD), and 10 mM EDTA. Proteasome inhibitor
complexes were generated by soaking crystals overnight in
reservoir buffer containing 1 mM compound, 10% DMSO and
20% MPD followed by an additional 5 hours in reservoir buffer
containing 1 mM compound, 10% DMSO and 25% MPD before
being flash-cooled in liquid nitrogen. Crystal data and refinement
statistics are given in the ESI Table†.
Biological assays
NFkB reporter assay. Cells were seeded at 10 000 per well in
white BioCoatꢀ poly-D-lysine (PDL)-coated 384-well plates (BD
Biosciences) 16–24 h prior to compound treatment. HEK293 cells
were pre-treated for 1 h with proteasome inhibitor and then
stimulated with 10 ng mLꢁ1 human recombinant tumor necrosis
factor (rhTNF)-a (R&D Systems) for a further 3 h in the continued
presence of the compound. Firefly luciferase activity was measured
using Bright-Gloꢀ reagents according to the manufacturer’s
instructions (Promega) in a LEADseekerꢀ plate reader (GE
Healthcare Life Sciences). Inhibition of NFkB-Luc activity was
calculated relative to a no-compound (DMSO) control.
Acknowledgements
We are grateful to Juan Gutierrez and Zhi Li their assistance with
preparation of the yeast 20S enzymes and to Abe Achab, David
Lok, Nina Molchanova, Mingkun Fu and Ashok Patil for
analytical chemistry assistance.
Cell viability assay. Calu6 cells (2000 cells per well) were plated
in black/clear-bottom BioCoatꢀ poly-D-lysine (PDL)-coated
384-well plates (BD Biosciences) and were incubated with
compound for 72 h, after which medium was removed to leave 25
mL per well. An equal volume of ATPliteꢀ reagent (Perki-
nElmer) was then added and luminescence was measured using
a LEADseekerꢀ instrument.
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This journal is ª The Royal Society of Chemistry 2012
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