New betulinic acid derivatives as potent proteasome inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.07.072

In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC₅₀ values of 1.42, 1.56, and 1.80 μM, respectively, which are three to fourfold more potent than the proteasome inhibition controls LLM-F and lactacystin.

Full text of DOI:10.1016/j.bmcl.2011.07.072

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5944–5947
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New betulinic acid derivatives as potent proteasome inhibitors
Keduo Qian a, , Sang-Yong Kim a, Hsin-Yi Hung a, Li Huang b, Chin-Ho Chen b, Kuo-Hsiung Lee a,c,
⇑
⇑
a Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
b Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of
the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and
C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipo-
philic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds
Received 9 June 2011
Revised 19 July 2011
Accepted 20 July 2011
Available online 26 July 2011
13, 20, and 21 showed the best proteasome inhibition activity with IC50 values of 1.42, 1.56, and 1.80 lM,
respectively, which are three to fourfold more potent than the proteasome inhibition controls LLM-F and
lactacystin.
Keywords:
Betulinic acid derivatives
Proteasome inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Betulinic acid (BA, 3b-hydroxy-lup-20(29)-en-28-oic acid, 1) is a
pentacyclic triterpene present in many plant species, for example,
Triphyophyllum peltatum, Ancistrocladus heyneanus, Ziziphi fructus,
Diospyros leucomelas, Tetracera boliviana and Syzygium formosa-
num,1–3 and can be obtained in quantity from the bark of the Lon-
don plane tree, Platanus acerifolia.4–6 BA (1) exhibits diverse
biological activities including anti-retroviral, anti-bacterial, anti-
malarial, anti-inflammatory, anti-cancer, anti-oxidant, and anthel-
mintic properties. Our previous research on 1 resulted in the
discovery of bevirimat [DSB, 3-O-(30,30-dimethylsuccinyl)-betulinic
acid, 2] (Fig. 1),7 which exhibits remarkable anti-HIV-1 activity with
a mean EC50 value of <10 nM against primary and drug-resistant
HIV-1 isolates,7,8 representing a unique first in a class of anti-HIV
compounds termed maturation inhibitors (MIs).8,9 Bevirimat has
recently succeeded in Phase IIb clinical trials.6,10–12 In the further
evaluation of 2, we discovered that HIV-1 virions produced in the
presence of 2 contained higher amounts of the cellular protein APO-
BEC3G, which is usually degraded by cellular proteasomes. We then
postulated that the elevated APOBEC3G level in these viral infected
cells may be due to the inhibition of proteasomes by 2.
catalytic. The b subunits of proteasomes have three major proteo-
lytic activities: chymotrypsin-like (b5), trypsin-like (b2), and cas-
pase-like (b1). These proteolytic activities enable the proteasome
to cleave proteins into small peptides. Proteasomes are involved
in many essential cellular functions, such as cell cycle regulation,
cell differentiation, signal transduction pathways, antigen process-
ing for appropriate immune responses, stress signaling, inflamma-
tory responses, and apoptosis. Targeting the proteasome-ubiquitin
pathway has thus been considered a novel strategy for the treat-
ment of various disorders such as neurodegenerative diseases, can-
cers, and inflammatory diseases.14,15
In our initial screening, we found that 2 inhibited the chymo-
trypsin-like activity of 20S proteasome with an IC50 value of
9.2
l
M, but it did not inhibit the caspase-like activity, and had a
M) on the trypsin-like
very weak inhibitory effect (IC50: 26.29
l
activity.16 These results are favorable because normal cells may
suffer fewer consequences from treatment with 2, as two of the
main proteolytic activities of proteasomes are retained. Indeed,
the anticancer drug bortezomib (PS341), the first successful pro-
teasome inhibitor for the treatment of multiple myeloma, also
showed differential inhibition of the three catalytic activities of
proteasomes, which is believed to be critical for its clinical benefit
in disease treatment.14,17,18 In addition, compound 2 did not inhibit
the cellular proteases cathepsin B (cysteine protease) and DPPIV
(DPP4, CD26, serine protease) in our screening.16 Consequently, it
may have reduced side effects compared with other proteasome
inhibitors such as peptide aldehydes and peptide boronates, which
also inhibit these cellular proteases. Therefore, to better establish a
structure–activity relationship (SAR) of the proteasome regulatory
activity of BA derivatives, diverse substituted BA analogs were de-
signed. This paper reports herein the synthesis and proteasome
inhibitory activity of these BA derivatives.
Proteasomes are responsible for more than 80% of intracellular
protein degradation.13 The most common form of the proteasome
is known as 26S proteasome, which contains one 20S core particle
structure and two 19S regulatory caps. All 20S particles consist of
four stacked heptameric ring structures that are themselves com-
posed of two different types of subunits,
a and b. Alpha subunits
are structural in nature, whereas b subunits are predominantly
⇑
Corresponding authors. Tel.: +1 919 966 1394 (K.Q.); tel.: +1 919 962 0066; fax:
+1 919 966 3893 (K.-H.L.).
E-mail addresses: kdqian@unc.edu (K. Qian), khlee@unc.edu (K.-H. Lee).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.072

K. Qian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5944–5947
5945
30
20
29
H
H
H
19
O
R
28
H
O
H
H
OH
O
O
OH
H
H
H
HO
3
O
HO
H
HO
H
O
H
N
H
O
OH
23
LH15bh (
IC9564 (
)
R=
R=
1
)
2
)
Betulinic Acid(BA,
Bevirimat(DSB,
O
H
N
24
)
(CH2)
7
N
H
COOH
OH
Figure 1. Structures of BA (1) and its derivatives (2, 23, 24).
O
O
O
O
O
MeO
MeO
MeO
7
11
HO
HO
3
: R=
: R=
: R=
MeO
OMe
H
H
O
3
a for
Br
O
O
O
H
COOH
H
COOH
8: R=
9: R=
4: R=
5: R=
6: R=
O
12: R=
13: R=
4 10, 13
b for
c for
-
H
O
H
11 12
,
OMeO
O
HO
RO
H
H
1
3-13
HO
MeO
Br
Br
O
O
OMe
O
O
10: R=
Scheme 1. Reactions and conditions: (a) acid chloride, Et3N, CH2Cl2, rt; (b) acid, EDCI, DMAP, Et3N, CH2Cl2, rt; (c) acid anhydride, DMAP, pyridine, reflux.
Because 2 is a C-3 modified BA derivative, we first synthesized
eleven 3-O-acyl-BA analogs (3–13) to investigate the impacts of dif-
ferent C-3 modifications on the proteasome inhibitory activity of BA
derivatives. As shown in Scheme 1 and 3,4,5-trimethoxybenzoyl
chloride was reacted with 1 in the presence of Et3N in CH2Cl2 at
room temperature to yield compound 3. Diverse carboxylic acids
were first treated with EDCI and DMAP in CH2Cl2 for 1 h, before
compound 1 and Et3N were added into the system. The mixtures
were stirred at room temperature to yield compounds 4–10 and
13. 1,2,3,6-Tetrahydrophthalic and diglycolic anhydrides were re-
fluxed with 1 in the presence of DMAP in pyridine to provide com-
pounds 11 and 12, respectively. To investigate the impact of 28- and
30-subsitutions on BA’s proteasome inhibitory effects, nine 28,30-
disubstituted BA analogs (14–22) were synthesized by the method
reported previously (Scheme 2)19 and were also tested against 20S
proteasome. Briefly, BA was first protected with acetic anhydride to
yield 25, which was treated with oxalyl chloride and then reacted
with leucine methyl ester to furnish 26. Allylic bromination of 26
was carried out using NBS in dilute acetonitrile to provide 27.
Saponification of 27 yielded 14. Meanwhile, nucleophilic displace-
ment of the bromide of 27 was conducted by treating varied nucle-
ophilic compounds with NaH, followed by their reaction with 27 in
a microwave apparatus at 120 °C. Saponification of the resulting
mixtures in MeOH–H2O converted the intermediate esters to car-
boxylic acids to yield the target compounds 15–22. Two com-
pounds with only a C-28 side chain, LH15bh (23) and IC9564 (24)
(Fig. 1),20,21 were also tested for their proteasome inhibition activity
to investigate the influence of C-28 mono-substitution.
substituted benzoate ester side chain at the C-3 position showed
moderate inhibition activity (IC50: 11.06 and 10.91 M, respec-
l
tively) on the chymotrypsin-like activity of 20S proteasome.
Compounds 3, 4, and 7 without ortho-methoxy substitution on ben-
zoate ester side chain abolished or greatly decreased the protea-
some inhibition activity. In a comparison of three compounds
with different brominated benzoate side chains, bromide on the
20- or 30-position (8 and 10) was advantageous (IC50: 7.99 and
7.05 l
M, respectively), while bromide on the 40-position (9) was
detrimental. Interestingly, while compound 12, with a polar oxygen
atom in the middle of a diglycolic ester side chain was inactive,
compounds with both a cyclohexene ring (11) and a phenyl ring
(13), located centrally within a dicarboxylic side chain were signif-
icantly active. Compound 11, with a 1,2,3,6-tetrahydrophthlate es-
ter side chain, exhibited increased proteasome inhibition activity
with an IC50 value of 2.96
30-acetylphenylacetate (13) further increased the proteasome inhi-
bition activity to an IC50 value of 1.42 M, which is sixfold better
lM. Elongation of the C-3 side chain to
l
than 2 and fourfold better than LLM-F and lactacystin. Among the
synthesized 3-O-acyl BA analogs, compound 13 showed the best
activity, suggesting that an aromatic lipophilic ester side chain with
a carboxylic acid terminus at the C-3 position can significantly in-
crease the proteasome inhibition activity of BA derivatives.
With respect to the effects of 28- and 30-substitutions on the
proteasome activity, we found that all nine 28,30-disubstituted
BA analogs (14–22) showed inhibitory activity towards the chymo-
trypsin-like activity of 20S proteasome. However, the 28-substi-
tuted BA derivatives 23 and 24 did not inhibit the 20S
proteasome to any degree. We therefore concluded that the protea-
some inhibition activity of the 28,30-disubstituted BA compounds
comes mainly from the contribution of the C-30 substituents.
Within the C-30 side chains, larger ether substituents (16–22) were
favored compared with bromide (14) or a smaller ethoxy group
(15). Among 16–22, the rank order of potency based on the C-30
ether substituent was 40-bromophenethoxy (20) = 40-chlorophe-
nethoxy (21) > 40-fluorophenethoxy (19) = phenethoxy (17) > pro-
poxy (16) = 40-methyoxyphenethoxy (18) = 20-morpholinoethoxy
(22). Overall, compound 15 with an ethoxy substituent showed
Table 1 lists the results for inhibition of the chymotrypsin-like
activity of 20S proteasome shown by BA derivatives 3–2422 to-
gether with compound 2. Known proteasome inhibitors Ac-Leu-
Leu-Met-CHO (LLM-F) and lactacystin were also included in the
in vitro 20S proteasome assay23 as controls. From the results with
compounds 2–13, we can conclude that addition of a C-3 ester
group to 1 can confer proteasome inhibition activity and the iden-
tity of the ester group can influence the potency. Comparing the
activity of 3–10, we found that compound 6 with an unsubstituted
benzoate ester side chain and compound 5 with 20,40,50-trimethoxy

5946
K. Qian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5944–5947
H
H
a
b
H
COOH
H
COOH
H
H
HO
AcO
H
H
25
1
Br
R1
O
O
H
H
O
H
H
N
H
N
H
N
c
e
H
OH
H
OMe
H
d
OMe
O
O
O
H
H
H
AcO
AcO
HO
H
H
H
15-22
26
27
15
OCH2CH3
R1
=
16 R1 =
O(CH2)2CH3
O(CH2)2
Br
17 R1
18 R1
19 R1
20 R1
21 R1
=
=
=
=
=
O
H
N
O(CH2)2
O(CH2)2
O(CH2)2
O(CH2)2
OMe
F
H
OH
O
H
HO
H
14
Br
Cl
22 R1
=
O
O(CH2)2
N
Scheme 2. Reactions and conditions: (a) Ac2O, pyridine, 78 °C, 4 h; (b) (CO2)Cl2, leucine methyl ester, Et3N, CH2Cl2, rt; (c) NBS, acetonitrile, rt; (d) 2 N NaOH, MeOH/THF, rt; (e)
step 1: nucleophilic compounds, NaH, THF; step 2: MeOH–H2O.
Table 1
proteasome inhibitors might be able to provide new therapeutic
agents for treating cancers and inflammatory diseases.
The inhibition on the chymotrypsin-like activity of 20S proteasome of 3–24 with 2,
LLM-F, and lactacystin as controls
a
a
Compound
IC50
(
lM)
Compound
IC50 (lM)
Acknowledgments
3
4
5
6
7
8
9
10
11
12
13
14
15
>40
>40
10.91
11.06
>40
7.99
>40
7.05
2.96
>40
16
17
18
19
20
21
22
23
3.67
2.47
3.34
2.40
1.56
1.80
3.13
> 40
> 40
9.20
5.25
5.60
This investigation was supported by Grant GM-084337 from
the National Instituted of General Medical Sciences (NIGMS)
awarded to C.H.C and AI-077417 from the National Institute of Al-
lergy and Infectious Diseases (NIAID) awarded to K.H.L. This study
was also supported in part by Taiwan Department of Health
Clinical Trial and Research Center of Excellence (DOH100-TD-B-
111-004). Efficient purification of all the synthetic BA analogs
was performed with a Grace RevelerisÒ flash chromatography sys-
tem equipped with RevealX™ detection allowing for multisignal
(UV/ELSD) collection to low mg quantities. RevelerisÒ Navigator
method optimizer and Grace RevelerisÒ flash silica cartridges were
employed for high quality separations.
24
2
1.42
5.85
13.21
LLM-F
lactacystin
a
The IC50 values in the table are the averages from three experiments.
the lowest activity among the 28,30-disubstituted BAs with an IC50
value of 13.21
M, while compounds 20 and 21 with 40-bromophe-
nethoxy and 40-chlorophenethoxy substituents, respectively,
showed the highest activity with IC50 values of 1.56 and 1.80 M,
respectively, threefold better than LLM-F and lactacystin. Thus,
longer C-30 side chains with aromatic substitutions were favored
for the proteasome inhibition activity.
In summary, this study showed that substituted BA analogs can
be proteasome inhibitors, and the C-3 and C-30 positions of BA are
the pharmacophores for improving the proteasome inhibition
activity. Within the C-3 and C-30 substitutions, larger side chains
with lipophilic or aromatic side chains are favored for increased
inhibition of the chymotrypsin-like activity of 20S proteasome.
C-3 and C-30 modified BA compounds showed low toxicity in
our previous anti-AIDS studies, and bevirimat (2), a C-3 substituted
BA, has already succeeded in phase IIb clinical trials. Therefore,
development of C-3 and/or C-30 modified BA derivatives as
References and notes
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each, s, 2 ꢀ CH3), 0.89, 0.83, 0.81 (3H each, s, 3 ꢀ CH3), MS m/z 607.40 (Mꢁꢁ1);
Compound 12: 1H NMR (300 MHz, CDCl3): d 4.72, 4.60 (1H each, s, H-29), 4.46
(1H, m, H-3), 4.23, 4.21 (2H each, s, H-20, 40), 3.01 (1H, m, H-19), 1.71 (3H, s, H-
30), 1.00, 0.98, 0. 79, 0.88, 0.87 (3H each, s, 5 ꢀ CH3), MS m/z 571.60 (Mꢁꢁ1);
1
Compound 13: H NMR (400 MHz, CDCl3): d 7.82 (1H, s, Ar-H-20), 7.57–7.50
(2H, m, Ar-H-40, 60), 7.30 (1H, m, Ar-H-50), 4.72, 4.60 (1H each, s, H-29), 4.40
(1H, m, H-3), 3.63, 3.60 (2H each, s, 2 ꢀ Ar-CH2–CO–), 2.98 (1H, m, H-19), 1.68
(3H, s, H-30), 0.95, 0.90, 0.82, 0.72, 0.68 (3H each, s, 5 ꢀ CH3), MS m/z 631.78
(Mꢁꢁ1); Compound 14: 1H NMR (300 MHz, CDCl3): d 5.86 (1H, d, J = 8.0 Hz, -
CONH-), 5.11, 5.02 (1H each, s, H-29), 4.65 (1H, m, –NHCH–), 3.90 (2H, s, H2-
30), 3.17 (1H, dd, J = 9.7, 5.4 Hz, H-3), 3.10–3.03 (1H, m, H-19), 1.00 (6H, s,
leucine moiety -(CH3)2), 0.96 (6H, s, 2 ꢀ CH3), 0.83, 0.80, 0.79 (3H each, s,
3 ꢀ CH3), MS m/z 646.41, 648.39 (Mꢁꢁ1); Compound 15: 1H NMR (300 MHz,
CDCl3): d 5.88 (1H, d, J = 8.0 Hz, –CONH–), 4.93, 4.92 (2H, br s, H-29), 4.63–4.58
(1H, m, –NHCH–), 3.90 (2H, s, H2-30), 3.47 (2H, m, 30-OCH2CH3), 3.18 (1H, dd,
J = 11.1, 5.4 Hz, H-3), 2.99 (1H, m, H-19), 2.50–2.32 (1H, m, H-13), 1.00 (9H, br
s, 30-OCH2CH3, leucine moiety –(CH3)2), 0.96 (6H, s, 2 ꢀ CH3), 0.89, 0.86, 0.85
(3H each, s, 3 ꢀ CH3), MS m/z 612.40 (Mꢁꢁ1); Compound 16: 1H NMR
(300 MHz, CDCl3): d 6.13 (1H, br s, –CONH–), 4.91, 4.90 (2H, br s, H-29), 4.52
(1H, m, –NHCH–), 3.90 (2H, s, H2-30), 3.36 (2H, t, J = 6.9 Hz, 30-OCH2CH2CH3),
3.18 (1H, dd, J = 11.1, 5.4 Hz, H-3), 2.99 (1H, m, H-19), 0.96, 0.94, 0.92, 0.89
(15H, m, 30-O(CH2)2CH3, leucine moiety –(CH3)2, CH3-23, 24), 0.82, 0.81, 0.79
(3H each, s, 3 ꢀ CH3), MS m/z 626.50 (Mꢁꢁ1); Compound 17: 1H NMR
(300 MHz, CDCl3): d 7.68–7.62 (2H, m, Ar-H-30), 7.28–7.20 (3H, m, Ar-H-20,
40), 5.97 (1H, br s, –CONH–), 4.91, 4.90 (2H, br s, H-29), 4.44 (1H, m, –NHCH–),
3.93 (2H, s, H2-30), 3.64 (2H, t, J = 7.2 Hz, 30-OCH2CH2Ph), 3.17 (1H, dd, J = 11.1,
5.4 Hz, H-3), 2.91 (1H, m, H-19), 2.57 (2H, m, 30-OCH2CH2Ph), 0.95 (12H, s,
leucine moiety –(CH3)2, CH3-23, 24), 0.89, 0.78, 0.74 (3H each, s, 3 ꢀ CH3), MS
m/z 688.41 (Mꢁꢁ1); Compound 18: 1H NMR (300 MHz, CDCl3): d 7.27, 7.16–
7.13, 6.85–6.82 (4H, m, Ar-H-20, 30), 5.97 (1H, br s, –CONH–), 4.91, 4.89 (H each,
br s, H-29), 4.48 (1H, m, –NHCH–), 3.93 (2H, s, H2-30), 3.79 (3H, s, Ar-OCH3),
3.60 (2H, t, J = 7.2 Hz, 30-OCH2CH2Ph(p-OCH3)), 3.17 (1H, dd, J = 11.1, 5.4 Hz, H-
3), 2.85 (1H, t, J = 7.5 Hz, H-19), 2.39 (3H, m, 30-OCH2CH2Ph(p-OCH3), H-13),
0.95, 0.93, 0.90 (15H, s, leucine moiety –(CH3)2, 3 ꢀ CH3), 0.79, 0.75 (3H each, s,
2 ꢀ CH3), MS m/z 718.50 (Mꢁꢁ1); Compound 19: 1H NMR (300 MHz, CDCl3): d
7.16–6.80 (4H, m, Ar-H-20, 30), 5.96 (1H, br s, -CONH-), 4.90, 4.89 (2H, br s, H-
29), 4.48 (1H, m, –NHCH–), 3.92 (2H, s, H2-30), 3.61 (2H, t, J = 7.2 Hz, 30-
OCH2CH2Ph(p-F)), 3.17 (1H, m, H-3), 2.87 (1H, t, J = 7.5 Hz, H-19), 2.36–2.10
(3H, m, 30-OCH2CH2Ph(p-F), H-13), 0.95, 0.88 (15H, s, leucine moiety –(CH3)2,
3 ꢀ CH3), 0.75, 0.73 (3H each, s, 2 ꢀ CH3), MS m/z 706.40 (Mꢁꢁ1); Compound
20: 1H NMR (300 MHz, CDCl3): d 7.56–7.28 (4H, m, Ar-H-20, 30), 5.96 (1H, br s, –
CONH–), 4.91, 4.90 (2H, br s, H-29), 4.48 (1H, m, –NHCH–), 3.91 (2H, s, H2-30),
3.60 (2H, t, J = 7.0 Hz, 30-OCH2CH2Ph(p-Br)), 3.17 (1H, dd, J = 11.0, 5.6 Hz, H-3),
2.89 (1H, t, J = 7.5 Hz, H-19), 2.39 (1H, m, 30-OCH2CH2Ph(p-Br)), 0.96 (12H, s,
leucine moiety –(CH3)2, 2 ꢀ CH3), 0.82, 0.79, 0.75 (3H each, s, 3 ꢀ CH3), MS m/z
766.38 (Mꢁꢁ1); Compound 21: 1H NMR (300 MHz, CDCl3): d 7.18–6.87 (4H, m,
Ar-H-20, 30), 5.96 (1H, br s, -CONH-), 4.91, 4.90 (2H, br s, H-29), 4.48 (1H, m, –
NHCH–), 3.91 (2H, s, H2-30), 3.62 (2H, t, J = 6.8 Hz, 30-OCH2CH2Ph(p-Cl)), 3.17
(1H, dd, J = 11.0, 5.6 Hz, H-3), 2.87 (1H, t, J = 7.5 Hz, H-19), 2.36–2.06 (3H, m,
30-OCH2CH2Ph(p-Cl), H-13), 0.96 (15H, s, leucine moiety –(CH3)2, 3 ꢀ CH3),
0.81, 0.76 (3H each, s, 2 ꢀ CH3), MS m/z 722.40 (Mꢁꢁ1); Compound 22: 1H NMR
(300 MHz, CDCl3): d 5.61 (1H, d, J = 8 Hz, –CONH–), 4.92, 4.90 (H each, s, H-29),
4.59 (1H, m, –NHCH–), 3.94 (2H, s, H2-30), 3.72 (4H, m, –N(CH2CH2)2O), 3.58
(2H, t, J = 5.7 Hz, 30-OCH2CH2-morpholine), 3.18 (1H, dd, J = 11.4, 4.6 Hz, H-3),
3.01 (1H, m, H-19), 2.60 (2H, t, J = 5.4 Hz, 30-OCH2CH2-morpholine), 2.53 (4H,
m, –N(CH2CH2)2O), 1.00 (6H, s, leucine moiety –(CH3)2), 0.96 (6H, s, 2 ꢀ CH3),
0.89, 0.85, 0.80 (3H each, s, 3 ꢀ CH3), MS m/z 697.40 (Mꢁꢁ1).
13. Nencioni, A.; Grunebach, F.; Patrone, F.; Ballestrero, A.; Brossart, P. Leukemia
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22. Materials and methods: 1H NMR spectra were measured on a 300 or 400 MHz
Varian Gemini 2000 spectrometer using TMS as internal standard. The solvent
used was CDCl3. Mass spectra were measured on a Shimadzu LC-MS2010
instrument. Thin-layer chromatography (TLC) was performed on precoated
silica gel GF plates purchased from Merck, Inc. All other chemicals were
obtained from Aldrich, Inc. Fast purification of crude reaction mixtures was
performed on the Grace RevelerisÒ flash system by prescreening the crude
mixtures on TLC plates to select appropriate solvent pairs for purification.
Method parameters were automatically loaded when the RevelerisÒ cartridge
was inserted or when the RevelerisÒ Navigator method optimizer was
employed to predict high purity or fastest separations, but were adjusted
manually as desired. Crude samples were dissolved in the appropriate solvent
before being injected automatically via solid loading. Spectroscopic data for
target compounds are shown as follows. Compound 3: 1H NMR (400 MHz,
CDCl3): d 7.29, 7.27 (1H each, s, Ar-H), 4.76, 4.64 (1H each, s, H-29), 4.60 (1H, m,
H-3), 3.94 (3H, s, Ar-OCH3), 3.91 (6H, s, 2 ꢀ Ar-OCH3), 3.06 (1H, m, H-19), 1.71
(3H, s, H-30), 0.97, 0.90, 0.83, 0.76, 0.70 (3H each, s, 5 ꢀ CH3), MS m/z 649.50
(Mꢁꢁ1); Compound 4: 1H NMR (400 MHz, CDCl3): d 7.64 (1H, d, J = 8.0 Hz, Ar-
H-60), 7.45 (1H, s, Ar-H-20), 6.84 (1H, d, J = 8.0 Hz, Ar-H-50), 6.03 (2H, s, –
OCH2O–), 4.73, 4.60 (1H each, s, H-29), 4.65 (1H, m, H-3), 3.10 (1H, m, H-19),
1.70 (3H, s, H-30), 0.99, 0.97, 0.96, 0.90, 0.89 (3H each, s, 5 ꢀ CH3), MS m/z
603.40 (Mꢁꢁ1); Compound 5: 1H NMR (400 MHz, CDCl3): d 7.44 (1H, s, Ar-H-
60), 6.52 (1H, s, Ar-H-30), 4.75, 4.62 (1H each, s, H-29), 4.69 (1H, dd, J = 8.0,
4.0 Hz, H-3), 3.93, 3.88, 3.86 (3H each, s, 3 ꢀ Ar-OCH3), 3.01 (1H, m, H-19), 1.70
(3H, s, H-30), 1.00, 0.97, 0.96, 0.95, 0.89 (3H each, s, 5 ꢀ CH3), MS m/z 649.70
(Mꢁꢁ1); Compound 6: 1H NMR (400 MHz, CDCl3): d 8.04 (2H, d, J = 8.0 Hz, Ar-
H-20, 60), 7.55 (1H, m, Ar-H-40), 7.45 (1H, m, Ar-H-30, 50), 4.75, 4.62 (1H each, s,
H-29), 4.71 (1H, m, H-3), 3.02 (1H, m, H-19), 1.71 (3H, s, H-30), 1.00, 1.00, 0.96,
0.93, 0.91 (3H each, s, 5 ꢀ CH3), MS m/z 559.45 (Mꢁꢁ1); Compound 7: 1H NMR
(400 MHz, CDCl3): d 7.68 (1H, d, J = 4.0 Hz, Ar-H-60), 7.56 (1H, s, Ar-H-20), 6.88
(1H, d, J = 4.0 Hz, Ar-H-50), 4.75, 4.62 (1H each, s, H-29), 4.69 (1H, m, H-3), 3.96,
3.93 (3H each, s, 2 ꢀ Ar-OCH3), 3.02 (1H, m, H-19), 1.71 (3H, s, H-30), 1.00, 0.99,
0.96, 0.92, 0.90 (3H each, s, 5 ꢀ CH3), MS m/z 619.45 (Mꢁꢁ1); Compound 8: 1H
NMR (400 MHz, CDCl3): d 7.75 (1H, d, J = 8.0 Hz, Ar-H-60), 7.64 (1H, d, J = 8.0 Hz,
Ar-H-30), 7.37–7.28 (2H, m, Ar-H-40, 50), 4.78 (1H, m, H-3), 4.75, 4.62 (1H each,
s, H-29), 3.01 (1H, m, H-19), 1.71 (3H, s, H-30), 1.00, 0.97, 0.95, 0.94, 0.89 (3H
each, s, 5 ꢀ CH3), MS m/z 639.40 (M++1); Compound 9: 1H NMR (400 MHz,
CDCl3): d 7.89 (2H, d, J = 8.0 Hz, Ar-H-20, 60), 7.59 (2H, d, J = 8.0 Hz, Ar-H-30, 50),
4.73, 4.60 (1H each, s, H-29), 4.70 (1H, m, H-3), 3.02 (1H, m, H-19), 1.70 (3H, s,
H-30), 1.00, 0.99, 0.97, 0.91, 0.90 (3H each, s, 5 ꢀ CH3), MS m/z 637.65 (Mꢁꢁ1);
23. 20S proteasome assay: 20S proteasome assay kits were purchased from
Calbiochem, San Diego, CA. Measurement of the hydrolysis of the fluorogenic
substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence of the proteasome
activator PA28 (16 lg/mL) is used to detect the chymotrypsin-like activity of
1
Compound 10: H NMR (400 MHz, CDCl3): d 8.15 (1H, s, Ar-H-20), 7.95 (1H, d,
the human 20S proteasome. Fluorescence generated from the proteolytic
reaction in the presence of various concentrations of BA derivatives was
measured using a BioTek fluorometer (Winooski, Vermont). The 50% inhibitory
concentration (IC50) is defined as the inhibitor concentration that reduces the
J = 8.0 Hz, Ar-H-60), 7.67 (1H, d, J = 8.0 Hz, Ar-H-40), 7.31 (1H, m, Ar-H-50), 4.75,
4.62 (1H each, s, H-29), 4.71 (1H, m, H-3), 3.01 (1H, m, H-19), 1.71 (3H, s, H-30),
1.00, 0.99, 0.96, 0.92, 0.91 (3H each, s, 5 ꢀ CH3), MS m/z 637.50 (Mꢁꢁ1);
Compound 11: 1H NMR (300 MHz, CDCl3): d 5.70 (2H, m, H-50, 60), 4.72, 4.60 (1H
each, s, H-29), 4.48 (1H, m, H-3), 3.02 (1H, m, H-19), 2.83, 2.72 (1H each, m, H-
20, 30), 2.32–2.27 (4H, m, 2 ꢀ CH2, H-40, 70), 1.68 (3H, s, H-30), 1.01, 0.97 (3H
reaction rate by 50%. The velocity of reaction
(DRFU(360/460)/min) was
plotted against the log-concentration of the inhibitor to determine the IC50
.