Discovery of a novel dipeptidyl boronic acid proteasome inhibitor for the treatment of multiple myeloma and triple-negative breast cancer

Article abstract of DOI:10.1039/c8ob02668h

A series of novel dipeptidyl boronic acid compounds were designed, synthesized and biologically investigated for the inhibition of the β5 subunit of 20S proteasome and several compounds showed high activities with IC₅₀ values of less than 10 nM. Some of these compounds potently inhibited the multiple myeloma (MM) cancer cell lines with IC₅₀ values of less than 10 nM. It was reported that the inhibition of both β2 and β5 subunits strongly increased the cytotoxicity of proteasome inhibitors in solid tumor cells, so some of the compounds were evaluated for the inhibition of the β2 subunit and the solid tumor triple-negative breast cancer cell line MDA-MB-231. The results showed that three compounds were active for both the β2 subunit and the triple-negative breast cancer cell line MDA-MB-231. The in vivo pharmacokinetic results showed that compound 8t had good biological parameters for both ig and iv administrations. An in vivo pharmacodynamic experiment showed that compound 8t inhibited the β5 subunit in whole blood more greatly than the marketed MLN9708 with the same dose at different time periods. A pathological analysis indicated that the injection of compound 8t in the tumor of a triple-negative breast cancer xenograft mice model led to tumor cell necrosis, nucleus condensation, deep staining, cell fragmentation, dissolution and neutrophil infiltration compared with the control group. The data in hand showed that compound 8t might be an effective candidate for the treatment of both MM and triple-negative breast cancer.

Full text of DOI:10.1039/c8ob02668h

Organic &
Biomolecular Chemistry
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Discovery of a novel dipeptidyl boronic acid
proteasome inhibitor for the treatment of multiple
myeloma and triple-negative breast cancer†
Cite this: Org. Biomol. Chem., 2019,
17, 683
Meng Lei,*a Huayun Feng, a Enhe Bai,b Hui Zhou,b Jia Wang,c Yanru Qin,d
Haoyang Zhang,b Xueyuan Wang,b Zhaogang Liu,c Ou Hai,c Jia Liuc and
Yongqiang Zhu*b
A series of novel dipeptidyl boronic acid compounds were designed, synthesized and biologically investi-
gated for the inhibition of the β5 subunit of 20S proteasome and several compounds showed high activi-
ties with IC50 values of less than 10 nM. Some of these compounds potently inhibited the multiple
myeloma (MM) cancer cell lines with IC50 values of less than 10 nM. It was reported that the inhibition of
both β2 and β5 subunits strongly increased the cytotoxicity of proteasome inhibitors in solid tumor cells,
so some of the compounds were evaluated for the inhibition of the β2 subunit and the solid tumor triple-
negative breast cancer cell line MDA-MB-231. The results showed that three compounds were active for
both the β2 subunit and the triple-negative breast cancer cell line MDA-MB-231. The in vivo pharmacoki-
netic results showed that compound 8t had good biological parameters for both ig and iv administrations.
An in vivo pharmacodynamic experiment showed that compound 8t inhibited the β5 subunit in whole
blood more greatly than the marketed MLN9708 with the same dose at different time periods. A patho-
logical analysis indicated that the injection of compound 8t in the tumor of a triple-negative breast
cancer xenograft mice model led to tumor cell necrosis, nucleus condensation, deep staining, cell frag-
mentation, dissolution and neutrophil infiltration compared with the control group. The data in hand
showed that compound 8t might be an effective candidate for the treatment of both MM and triple-nega-
tive breast cancer.
Received 29th October 2018,
Accepted 13th December 2018
DOI: 10.1039/c8ob02668h
rsc.li/obc
dependent conversion of cellular proteins,5,6 and is composed
of 19S and 20S proteasomes.7
Introduction
The ubiquitin-proteasome pathway (UPP) is able to selectively
The 19S proteasome is composed of 18 subunits, which
degrade intracellular proteins and is involved in many physio- control the recognition, unfolding and translocation of the
logically important cellular processes including signal trans- protein substrates into the lumen of the 20S proteasome.8 The
duction, immune responses, DNA repair, unfolded protein 20S proteasome is composed of 28 protein subunits arranged
responses and cell cycle progression.1–4 The UPP and the in four stacked rings, with each ring comprising seven α- and
pathophysiology of cell activity have a close relationship and β-type subunits, following an α1–7β1–7 stoichiometry.9,10 The two
are of great significance for the maintenance of cell homeosta- outer chambers are formed by α subunits, while the central
sis. The 26S proteasome is a multi-catalytic protease complex chamber, containing proteolytic active sites, is made up of β
that relies on ATPase and is responsible for the ubiquitin- subunits. Three of the 14 β subunits are responsible for the
post-glutamyl peptide hydrolysis activity (PGPH, attributed to
β1), trypsin-like activity (T-L, β2), and chymotrypsin-like activity
aCollege of Science, Nanjing Forestry University, No. 159 Longpan Road, Nanjing
(CT-L, β5), respectively, and all these three active subunits
210037, PR China. E-mail: hk-lm@163.com
bCollege of Life Science, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing
hydrolyze the amide bond of protein substrates with the hydro-
philic γ-hydroxyl group of the N-terminal threonine (Oγ-Thr1).
Up to now, three proteasome inhibitors bortezomib, carfil-
zomib and MLN9708 (structures shown in Fig. 1) have been
approved by the FDA for the treatment of multiple myeloma
(MM) and the patients greatly benefited from these drugs.
These molecules could covalently bind the β5 subunit of 20S
210037, PR China. E-mail: zhyqscu@163.com
cJiangsu Chia Tai Fenghai Pharmaceutical Co. Ltd, No. 9 Weidi Road, Nanjing
210046, PR China
dSchool of Bio-engineering, Qilu University of Technology (Shandong Academy of
Sciences), No. 3501 Daxue Rd. Changqing District, Jinan, 250353, PR China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob02668h
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Results and discussion
Chemistry
The synthesis of amino acid methyl ester hydrochlorides 1a–1c
is illustrated in Scheme 1. Methyl esters 1a–1b were syn-
thesized from various amino acids using SOCl2 and directly
used in the next reaction without purification. 1c was prepared
from L-cysteine hydrochloride monohydrate which reacted with
MeI to give methyl substituted L-cysteine and then was esteri-
fied to produce 1c (yield 95.4%). Scheme 2 summarizes the
synthesis of the intermediate 2f with a total yield of 12.7%,
which was prepared according to the literature.24,25 Firstly, the
phthaloyl group reacted with ethyl chloroformate to form
amide 2a, which was ammonified by L-alanine under basic
Fig. 1 Structures of proteasome inhibitors approved by the FDA.
proteasome and induce cancer cell apoptosis. Although impor- conditions to give intermediate 2b with a yield of 79.3%.
tant therapeutic advances have been made, these drugs were Intermediate 2b then coupled with the directing group
associated with significant and dose-limiting off-target 8-aminoquinoline to obtain amide 2c with a yield of 71.9%.
toxicities11–14 and the development of acquired resistance.15,16 2,3-Dihydrobenzo[b][1,4]dioxine was introduced in the methyl
Despite the consistently high response rates with proteasome group of alanine via the palladium-catalyzed reaction to afford
inhibitor-based combinations, almost all MM patients even- the protected amino acid 2d with a yield of 66.7%. The
tually relapsed with progressively lower rates and durations of directing group 8-aminoquinoline of 2d was removed by the
response with each subsequent line of therapy and poor prog- treatment of BF3·Et2O in MeOH to afford the corresponding
nosis as resistance emerged.17
amino acid methyl ester 2e (yield 65.3%) with the amine pro-
Furthermore, no clinical cases have been reported that such tected by the phthaloyl group which was removed using
kinds of drugs were effective for solid tumors. Recently, ethylenediamine to afford unnatural amino acid methyl ester
Weyburne et al.18 reported that the inhibition of the activities 2f (yield 64.9%).
of both β2 and β5 subunits could strongly increase the anti-
The synthesis of target compounds 8a–8p and oral com-
neoplastic effects and the cytotoxicity of proteasome inhibitors pounds 8q–8t and MLN9708 is illustrated in Scheme 3.
in solid tumors. Rolfe19 further pointed out that the inhibition
of the β2 subunit would sensitize the β5 inhibitor to triple-
negative breast cancer cells.
Triple-negative breast cancer is the clinical manifestation of
an aggressive course. A number of clinical studies showed that
this type of breast cancer has the worst prognosis, there is a
high probability of brain metastasis and mortality, and there
are no effective drugs in the clinic.20–23 Consequently, there is
a medical need for the development of proteasome inhibitors
Scheme 1 Synthesis of amino acid methyl ester hydrochlorides 1a–1c.
which can be used for the treatment of both MM and triple-
negative breast cancer.
Reagents and conditions: (i) 1a–1b, SOCl2, MeOH, 0 °C; 1c, (1) 30%
sodium methoxide–methanol solution, MeI, 0 °C to rt; (2) SOCl2, MeOH,
−10 °C to rt.
In this study, a series of novel dipeptidyl boronic acid com-
pounds with variations of four different substitutions were
designed, synthesized and then biologically evaluated in vitro
and in vivo. The structure–activity relationship (SAR) was
summarized and the enzymatic inhibition results showed
that several compounds were active for both CT-L and T-L
activities and the cellular activities indicated that the com-
pounds were potent toward the MM and triple-negative breast
cancer cell lines. Injection of the candidate compound into
the tumors of a triple-negative breast cancer xenograft mice
model caused tumor cell necrosis, nucleus condensation,
deep staining, cell fragmentation, dissolution and neutrophil
infiltration, which might offer a new tool to fight triple-nega-
tive breast cancer. In vivo pharmacokinetic and pharmacody-
namic data further indicated that a candidate compound
could be developed for the treatment of MM and triple-nega-
tive breast cancer.
Scheme 2 Synthesis of unnatural amino acid methyl ester 2f. Reagents
and conditions: (i) ClCOOEt, Et3N, DMF, rt; (ii) L-alanine, Na2CO3, H2O,
rt; (iii) (1) SOCl2, CH2Cl2, 42 °C; (2) 8-aminoquinoline, DIPEA, CH2Cl2,
−20 °C to rt; (iv) 6-iodo-2,3-dihydrobenzo[b][1,4]dioxine, Pd(OAc)2,
AgBF4, t-BuOH, 85 °C; (v) BF3·Et2O, MeOH, 100 °C; (vi) ethylenediamine,
MeOH, 70 °C.
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Table 1 Structures of compounds 8a–8t, bortezomib, MLN2238, and
MLN9708 and proteasome inhibitory activities
a
IC50
Compd
R1
R2
H
R3
R4
(nM)
8a
H, H
11.2
Scheme 3 General synthesis of dipeptidyl boronic acids 8a–8p and
prodrugs 8q–8t. Reagents and conditions: (i) R1COOH, EDCI, HOBt,
DIPEA, CH2Cl2, −10 °C to rt; (ii) (1) LiOH·H2O, MeOH, H2O, rt; (2) 1N HCl,
ethyl acetate, rt; (iii) EDCI, HOBt, DIPEA, CH2Cl2, −10 °C to rt; (iv) (1)
EDCI, HOBt, DIPEA, CH2Cl2, −10 °C to rt; (2) HCl in ethyl acetate,
CH2Cl2, −5 °C to rt; (v) R1COCl, Et3N, CH2Cl2, −15 °C to rt, or R1COOH,
EDCI, HOBt, DIPEA, CH2Cl2, −10 °C to rt; (vi) isobutylboronic acid, 1 N
HCl, MeOH, hexane, rt; (vii) ethyl acetate, 74 °C to rt.
8b
8c
H, H
H, H
990
57.4
8d
8e
H, H
H, H
4.4
3.9
N-terminal protected acids of 4a–4j reacted with the commer-
cially available amino boronate trifluoroacetate 5a using the
common peptide synthesis method to afford the dipeptidyl
boronic acid pinanediol esters. Then the N-terminal protected
group Boc was removed with HCl in ethyl acetate to form
boronic acid ester hydrochlorides 6a–6e (yields 66.2–89.7%).
Intermediates 6a and 6b reacted with 2-morpholinoacetyl
chloride in the presence of triethylamine (Et3N) to obtain the
corresponding peptide boronic acid esters 7a–7b (yields 39.7%
and 95.1%, respectively), while boronic acid ester hydrochlo-
rides 6c–6j were coupled with various acids R1COOH in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) to
afford the corresponding peptide boronic acid esters 7c–7j
(yields 41.8–81.6%).
Amino acid methyl esters 1a–1c and 2f were coupled with
various acids R1COOH in the presence of EDCI and HOBt to
afford the corresponding peptide esters, then saponified and
acidified to give amino acids 3a–3f (yields 84.5–90.9%). Acids
3a–3f reacted with the commercially available amino boronate
trifluoroacetates 5a–5c using the peptide synthesis method to
afford the dipeptidyl boronic acid esters 7k–7p (yields
60.6–85.5%).
8f
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
1.2
1.3
0.9
3.2
81.6
2.6
9.7
8.6
4.3
8g
8h
8i
8j
8k
8l
Finally, after the transesterification of 7a–7p with isobutyl-
boronic acid, target compounds 8a–8p were obtained in mod-
erate to high yields (27.9–86.4%). Dipeptidyl boronic acids
MLN2238 and bortezomib reacted with diethanolamine, tri-
ethanolamine, and citric acid, respectively, to afford the corres-
ponding prodrugs 8q–8t and MLN9708 (yields 53.3–85.4%).
The structures of the target compounds are displayed in
Table 1.
8m
8n
Biological evaluation
Enzyme. The capacities of the target compounds to inhibit
the CT-L activity of 20S human proteasome were assayed using
appropriate fluorogenic substrates. The marketed bortezomib,
MLN9708 and its active form MLN2238 were used as the stan-
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Table 1 (Contd.)
pyrazin-2-yl (bortezomib, 7.0 nM) were more active than mor-
pholine ethyl (8a, 11.2 nM). A comparison between the activi-
ties of 8b (990 nM) and MLN2238 (7.7 nM) further confirmed
the fact that the hydrophilic morpholine ethyl substituent at
the R1 position was not beneficial for the activity. For the ter-
tiary butyl groups at the R2 position, the activity order of
different substituents at the R1 position was as follows: 2,5-
dichlorobenzyl (8e, 3.9 nM) > 5,6,7,8-tetrahydronaphthalene-1-
yl (8d, 4.4 nM) > pyrazin-2-yl (8c, 57.4 nM). The data indicated
that hydrophobic 2,5-dichlorobenz-yl and 5,6,7,8-tetrahydro-
naphthalene-1-yl substituents were more beneficial for activi-
ties than the hydrophilic pyrazin-2-yl one, which was consist-
ent with our previous studies.26–28
a
IC50
Compd
R1
R2
R3
R4
(nM)
8o
H, H
NAb
8p
8q
H, H
414
8.5
The effects of fluoro substituents at the R2 position on the
biological activities were also investigated. Both monofluoro
(8f, 1.2 nM) and difluoro (8g, 1.3 nM) substituted benzyl
groups were nearly as active as phenyl 8h (0.9 nM) and more
active than compound 8k (2.6 nM), where a new six-membered
ring was formed between dioxane and the phenyl ring. The
biological results also showed that a different chiral carbon at
the R2 position greatly affected the inhibitory activities. It
turned out that (R)-isomer 8i (3.2 nM) was 25-fold more active
than its (S)-isomer 8j (81.6 nM).
8r
H
H
5.6
7.3
4.6
7.0
8s
Finally, replacement of the isobutyl group at the R3 position
of compound 8k (2.6 nM) with n-butyl (8o, NA) and 4-methyl
benzyl (8p, 414 nM) greatly reduced the activities of these com-
pounds, which suggested that isobutyl at this position was
beneficial for the activity.
To investigate the oral drug availability, four prodrugs (8q–
8t) were prepared and they showed similar activities to their
active forms and MLN9708, which indicated that they have
promising potential to be developed as oral drugs.
8t
Bortezomib
Cell. In this study, the compounds with IC50 values of enzy-
matic inhibition less than 10 nM were further investigated to
evaluate their potential antitumor activities in multiple
myeloma cancer cell lines RPMI8226, U266B and ARH77. The
cellular activities of target compounds bortezomib, MLN2238
and MLN9708 are displayed in Table 2.
MLN2238
MLN9708
7.7
8.4
Most compounds showed significant inhibitory potency
against multiple myeloma cell lines RPMI8226, ARH77 and
U266B, which was consistent with the enzymatic results. For
the R2 substituents, it indicated that aromatic groups were
nearly as active as the aliphatic ones, such as phenyl and 2-F
phenyl substituted compounds 8h and 8f and the isobutyl
group 8e. However, the hydrophobic fragment at the R1 posi-
tion was more beneficial for the inhibition of RPMI8226 cell
lines than the hydrophilic ones, such as the 2,5-dichlorobenzyl
a IC50 values obtained for bortezomib, MLN2238 and MLN9708 under
our experimental conditions. b NA: no activity.
dards. The IC50 values of the final molecules are shown in (8i, 97.0 nM) and pyrazin-2-yl (8m, 43.4 nM), while both com-
Table 1.
pounds showed high activities against ARH77 and U266B cell
Morpholine ethyl groups were employed at the R1 position lines (Table 2). Prodrug 8t, which was substituted with the
to improve the solubility of these compounds (8a and 8b). methylthio methene group at the R2 position also showed
With the morpholine ethyl fragments at the R1 position, the good activities against three cancer cell lines.
phenyl substituent at the R2 position (8a, 11.2 nM) was much
Currently, there are no effective methods for the treatment
more beneficial to the potency compared with the H atom at of triple-negative breast cancer in clinical trials, and protea-
the R2 position (8b, 990 nM). Furthermore, the aromatic 2,5- some inhibitors have not shown clinical efficacy on solid
dichlorobenzyl group at the R1 position (8h, 0.9 nM) and tumors. Recently, Weyburne et al.18 reported that the inhi-
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Table 2 The cellular activities of compounds against MM cell lines
(IC50, nM)
Pharmacokinetic studies. Compound 8t showed good activi-
ties both for enzymatic and cellular profiles, and with these
encouraging in vitro data, compound 8t was further investigated
for the ig and iv pharmacokinetics in male Sprague-Dawley (SD)
rats. The pharmacokinetics parameters are listed in Table 4.
The results illustrated that the elimination phase half-lives of
compound 8t were 2.41 h for ig and 2.08 h for iv. However, the
bioavailability of compound 8t was only 11.0% in this experi-
ment, which needs to be improved in the future modification.
Pharmacodynamics studies. Compound 8t was further used
for ig pharmacodynamics studies in the whole blood of male
Sprague-Dawley (SD) rats at a dose of 5 mg kg−1 to investigate
the effects on CT-L proteasome activity. Fig. 2 shows that com-
pound 8t inhibited the CT-L proteasome activity in whole
blood more greatly than MLN9708 at the same dose at
different time periods, which indicated that candidate 8t
might be more active than the marketed MLN9708 in vivo.
Nowadays the evaluation of in vivo antitumor efficacy using a
xenograft mouse model of RPMI8226 is being carried out. As
for the toxicity of compound 8t, no obvious loss of body
weight and other side effects were observed during the phar-
macokinetics and pharmacodynamics studies. The toxicities of
compound 8t were systematically evaluated.
Compd
RPMI8226
U266B
ARH77
8d
8e
8f
8g
8h
8i
8k
8l
8m
8q
8r
8s
181.4
69.7
23.1
25.16
23.1
97.0
17.52
41.1
43.4
11.3
64.33
43.4
8.9
48.6
20.8
24.1
28.29
25.9
9.4
9.15
65.8
6.4
10.6
23.3
34.3
58.8
11.3
52.5
67.0
32.2
21.0
34.7
NTa
28.3
10.0
10.20
69.4
8.8
7.9
19.1
28.6
35.2
9.7
65.0
43.5
8t
Bortezomib
MLN2238
MLN9708
11.0
55.2
49.4
a NT: not tested.
bition of the β2 subunit could sensitize the β5 inhibitor to
triple-negative breast cancer cells. So in this study, we also
evaluated the inhibition activities of some potent compounds
toward the β2 subunit and the cytotoxic potential against the
triple-negative breast cancer cell line MDA-MB-231. The results
are shown in Table 3.
In vivo antitumor activity. Since compound 8t showed nano-
mole scale activity against the triple-negative breast tumor
The data indicated that some of the compounds had sig-
nificant inhibitory effects on triple-negative breast cancer cells.
Particularly compounds 8k, 8l, 8q and 8t showed the best
activities on the MDA-MB-231 cell line and compounds 8k, 8q
and 8t showed good activities on the β2 subunit. However, the
results of microsomal stabilities showed that compounds 8k,
8l and 8q metabolized more rapidly than 8t (data not shown),
so prodrug 8t was further developed as a candidate. The dis-
covery broadened the therapeutic areas of dipeptide boronic
acid proteasome inhibitors, and even provided a new way for
the treatment of breast cancer.
Fig. 2 The inhibition of CT-L proteasome activities in the whole blood
of SD rats with compounds MLN9708 and 8t.
Table 3 The proteasome inhibitory activities against the β2 subunit (IC50, nM) and the cellular activities against the triple-negative breast cancer
cell line MDA-MB-231 (IC50, nM)
Compd
β2
MDA-MB-231
Compd
β2
MDA-MB-231
8k
8l
8m
8n
498
882
8975
8690
3998
9.4
9.2
22.3
21.9
7.5
8q
8r
8s
8t
572
6.7
7623
8513
534
18.1
20.2
8.4
Bortezomib
MLN2238
7954
21.1
Table 4 Single dose ig and iv pharmacokinetic profiles of 8t in SD rats
Compd
Dose (mg kg−1
)
T1/2 (h)
AUClast (h ng mL−1
)
MRTlast (h)
Vz_obs (mL kg−1
)
F (%)
8t
iga
ivb
2
2
2.41
2.08
208
1890
2.35
1.55
—
2914
11.0
a ig: intragastric administration. b iv: intravenous injection.
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periods. Pathological analysis indicated that the injection of
compound 8t into the tumor was effective in the case of the
triple-negative breast cancer xenograft mice model compared
with the control group, which showed that compound 8t could
be a candidate drug for the treatment of triple-negative breast
cancer.
Fig. 3 Pathological analysis of the triple-negative breast tumor tissues
treated with solvent (A) and compound 8t (B).
Experimental
Chemistry
cells MDA-MB-231 (Table 3), we tried to investigate the in vivo Unless otherwise stated, commercial reagents were used
activity of compound 8t in the treatment of xenograft nude directly without any purification, and all solvents were dried
mice inoculated with triple-negative breast tumor cells by standard methods before use. Yields refer to chromato-
MDA-MB-231. Due to the low oral bioavailability of compound graphically obtained results and were not optimized unless
8t, it was injected directly in the tumors of nude mice at a otherwise stated. Reactions were monitored by thin-layer
dose of 1.15 mg kg−1. During the experiment, no shrinkage of chromatography on precoated silica gel GF254 plates and the
the tumor volume was observed. However, the tumors of the spots were tested under 254 nm UV light, 20% n-butyl alco-
treatment group became much softer than those of the control holic ninhydrin, 10% ethanolic phosphomolybdic acid and
group. After the experiment was finished, pathological analysis heat as a developing agent. HPLC showed that the purity of all
of the tumor tissues was performed and the results are shown the final products was greater than 95%. Melting points were
in Fig. 3.
determined on an YRT-3 melting point apparatus. 1H NMR
As can be seen from Fig. 3, only focal tumor cell necrosis, and 13C NMR spectra were recorded at room temperature on a
nucleus condensation, deep staining, fragmentation and dis- Bruker ARX400 spectrometer using TMS as the internal stan-
solution was observed in the control group (indicated by the dard. Chemical shifts are reported in ppm (δ units) and coup-
black arrow, A). However, after the treatment with compound ling constants (J) are expressed in hertz. The following abbrevi-
8t, many tumor cells showed necrosis, nucleus condensation, ations were used to designate the multiplicities as s (singlet), d
deep staining, cell fragmentation, dissolution and neutrophil (doublet), t (triplet), q (quartet), dd (doublet of doublets) and
infiltration (indicated by the black arrow, B). The results indi- m (multiplet). Mass spectra were recorded on Thermo Fisher
cated that compound 8t has promising potential for the treat- LC-MS instruments in electrospray positive and negative
ment of triple-negative breast tumors.
ionization modes. High-resolution mass spectra (HRMS) were
recorded on a ZAB-HS instrument using an electrospray source
(ESI).
(S)-Methyl 2-amino-2-cyclohexylacetate hydrochloride (1a).
To a solution of L-cyclohexylglycine (0.5 g, 3.18 mmol) dis-
Conclusions
A series of novel dipeptidyl boronic acid proteasome inhibitors solved in MeOH (5 mL) was added SOCl2 (2.3 mL, 31.8 mmol)
were designed, synthesized and biologically investigated dropwise at −10 °C. The mixture was stirred at room tempera-
in vitro and in vivo by varying different substituents at the R1, ture overnight. After evaporation, the obtained product was
R2, R3 and R4 positions of the backbone. The screening of the directly used in the next reaction.
inhibition of CT-L activities and MM cancer cell lines showed
(R)-Methyl 2-amino-2-cyclohexylacetate hydrochloride (1b). A
that several compounds were active. To investigate whether similar method was used for 1a with the corresponding start-
this kind of proteasome inhibitor was active for the treatment ing materials.
of solid tumors, we carried out the inhibition of the β2
(S)-Methyl 2-amino-3-(methylthio)propanoate hydrochloride
subunit and the triple-negative breast cancer cell line (1c). To a solution of L-cysteine hydrochloride monohydrate
MDA-MB-231. The results indicated that several compounds (3.1 g, 17.5 mmol) in anhydrous methanol (45 mL) was drop-
could inhibit the activities of the β2 subunit and the cancer wise added 30% sodium methoxide–methanol solution
cell line with IC50 values of less than 0.6 μM and 10 nM, (11 mL) at 0 °C. The reaction mixture was stirred for 1 h and
respectively, which illustrated that these compounds might be methyl iodide (0.9 mL, 13 mmol) was added dropwise. The
effective for the treatment of triple-negative breast cancer. mixture was allowed to react at room temperature for 2 h. The
Among the screened compounds, candidate 8t exhibited good solution was acidified with aqueous HCl (1 N) until pH = 5,
pharmacokinetic properties with half-lives of 2.41 h for ig and and then diethyl ether (40 mL) was added, affording a cloudy
2.08 h for iv. However, the oral bioavailability of 8t was only white suspension. After evaporation, the solid was washed
11%, which needs to be improved in the future work. The with ether (60 mL) and dried under vacuum to give a crude
pharmacodynamic data of the whole blood of the SD rats product. To a solution of the crude product in anhydrous
showed that compound 8t inhibited the CT-L activity more methanol (25 mL) was dropwise added SOCl2 (25 mL) at
greatly than MLN9708 with the same dose at different time −10 °C. The mixture was stirred for 10 min and then allowed
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to react at room temperature overnight. After evaporation, the evaporation and purification by chromatography (petroleum
solid was washed with CH2Cl2 (50 mL) and dried under ether/EtOAc = 3 : 1), 0.38 g (73.1%) of glassy solid was
vacuum to give 1c (3.1 g, 95.4%). 1H NMR (400 MHz, D2O) obtained. 1H NMR (400 MHz, CDCl3) δ 0.85 (d, J = 14.8 Hz,
δ 2.18 (s, 3H), 3.07–3.14 (m, 1H), 3.23 (dd, J1 = 4.6 Hz, J2
15.1 Hz, 1H), 3.90 (s, 3H), 4.44 (dd, J1 = 4.6 Hz, J2 = 7.7 Hz, 1.28–1.34 (m, 1H), 1.37 (s, 3H), 1.43–1.51 (m, 1H), 1.52–1.63
1H). MS (ESI) m/z 150.2 [M + H]+.
(m, 1H), 1.65 (d, J = 5.7 Hz, 1H), 1.77 (d, J = 11.0 Hz, 5H), 1.83
=
3H), 0.87–0.93 (m, 6H), 1.00–1.23 (m, 5H), 1.27 (s, 3H),
(S)-2-Cyclohexyl-2-(2,5-dichlorobenzamido)acetic acid (3a). (d, J = 11.1 Hz, 2H), 1.88 (s, 1H), 2.00 (dd, J1 = 5.8 Hz, J2 = 11.2
To a cooled solution (−10 °C) of 2,5-dichlorobenzoic acid Hz, 1H), 2.16 (dd, J1 = 5.1 Hz, J2 = 10.7 Hz, 1H), 2.26–2.36 (m,
(0.6 g, 3.4 mmol) dissolved in anhydrous CH2Cl2 (110 mL) was 1H), 3.15–3.27 (m, 1H), 4.29 (dd, J1 = 1.8 Hz, J2 = 8.7 Hz, 1H),
added HOBt (0.9 g, 6.9 mmol). After 10 min, EDCI (1.3 g, 4.47–4.54 (m, 1H), 6.42–6.58 (m, 1H), 6.86–7.06 (m, 1H), 7.32
6.9 mmol) was added. Finally, 1a (0.7 g, 3.4 mmol) and DIPEA (s, 2H), 7.55 (d, J = 1.6 Hz, 1H). MS (ESI) m/z 577.4 [M + H]+.
(1.8 g, 13.8 mmol) were added. The mixture was stirred at
Compounds 7j and 7n were prepared following a similar
−10 °C for 1 h and at room temperature for 15 h. The mixture procedure described for the synthesis of 7i.
was washed with 10% hydrochloric acid, 5% NaHCO3 and
2,5-Dichloro-N-((R)-1-cyclohexyl-2-(((R)-3-methyl-1-((3aS,4S,6S,
brine, respectively, and dried over anhydrous Na2SO4. After fil- 7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]
tration and evaporation, the obtained crude product was dioxaborol-2-yl)butyl)amino)-2-oxoethyl)benzamide (7j). A
1
directly used in the next reaction.
glassy solid (0.32 g, 60.6%). H NMR (400 MHz, CDCl3) δ 0.83
The prepared methyl ester was dissolved in methanol (s, 3H), 0.90 (dd, J1 = 3.2 Hz, J2 = 6.5 Hz, 6H), 1.02–1.23 (m,
(10 mL) and LiOH·H2O (0.9 g, 8.85 mmol) and H2O (3 mL) 5H), 1.27 (s, 3H), 1.30–1.35 (m, 1H), 1.37 (s, 3H), 1.41–1.46 (m,
were added. The mixture was stirred for 3 h at room tempera- 1H), 1.46–1.52 (m, 1H), 1.62–1.66 (m, 1H), 1.71–1.84 (m, 5H),
ture and the methanol was evaporated under vacuum. The 1.86 (dd, J1 = 3.3 Hz, J2 = 5.4 Hz, 2H), 1.89 (dd, J1 = 2.6 Hz, J2 =
aqueous phase was extracted with diethyl ether (5 mL) twice. 5.4 Hz, 1H), 2.02 (t, J = 5.5 Hz, 1H), 2.13–2.20 (m, 1H),
Then the aqueous phase was acidified with 1 N HCl until the 2.28–2.38 (m, 1H), 3.21 (dd, J1 = 5.9 Hz, J2 = 9.2 Hz, 1H), 4.30
pH was 2. The product was extracted with ethyl acetate and (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1H), 4.46 (dd, J1 = 7.1 Hz, J2 = 8.6
dried over anhydrous Na2SO4. After filtration and evaporation, Hz, 1H), 6.32 (d, J = 5.5 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H),
0.9 g (85.2%) of white solid was obtained. Mp 158.7–160.7 °C. 7.31–7.33 (m, 2H), 7.55 (dd, J1 = 1.1 Hz, J2 = 1.7 Hz, 1H). MS
1H NMR (400 MHz, CDCl3) δ 1.10–1.29 (m, 5H), 1.76 (dd, J1 = (ESI) m/z 577.4 [M + H]+.
12.9 Hz, J2 = 28.1 Hz, 5H), 1.96–2.02 (m, 1H), 4.78 (dd, J1
=
2,5-Dichloro-N-((R)-1-(((R)-3-methyl-1-((3aS,4S,6S,7aR)-3a,5,5-
4.8 Hz, J2 = 8.5 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 7.34 (t, J = trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-
5.0 Hz, 2H), 7.67 (dd, J1 = 4.3 Hz, J2 = 5.8 Hz, 1H). MS (ESI) m/z yl)butyl)amino)-3-(methylthio)-1-oxopropan-2-yl)benzamide
328.3 [M − H]−.
(7n). A glassy solid (2.1 g, 71.3%). 1H NMR (400 MHz, CDCl3) δ
Compounds 3b–3c were prepared from the corresponding 0.82 (d, J = 6.6 Hz, 3H), 0.92 (dd, J1 = 2.9 Hz, J2 = 6.6 Hz, 6H),
amino acids and carboxylic acids following a similar procedure 1.23 (dd, J1 = 5.9 Hz, J2 = 10.2 Hz, 2H), 1.28 (s, 3H), 1.38 (s,
described for the synthesis of 3a.
3H), 1.42–1.48 (m, 1H), 1.80–1.87 (m, 1H), 1.90 (dd, J1 =
(R)-2-Cyclohexyl-2-(2,5-dichlorobenzamido)acetic acid (3b). A 5.6 Hz, J2 = 8.2 Hz, 1H), 2.00–2.06 (m, 2H), 2.13–2.21 (m, 1H),
white solid (0.91 g, 87.5%). Mp 155.2–156.1 °C. 1H NMR 2.23 (s, 3H), 2.32 (dd, J1 = 7.8 Hz, J2 = 18.5 Hz, 1H), 2.82–2.88
(400 MHz, CDCl3) δ 1.09–1.31 (m, 5H), 1.78 (dd, J1 = 12.4 Hz, (m, 1H), 3.05 (dd, J1 = 4.6 Hz, J2 = 14.0 Hz, 1H), 3.27–3.42 (m,
J2 = 25.9 Hz, 5H), 1.98–2.05 (m, 1H), 4.80 (dd, J1 = 4.8 Hz, J2 = 1H), 4.29–4.33 (m, 1H), 4.65–4.76 (m, 1H), 6.64 (dd, J1
=
8.6 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 7.34–7.40 (m, 2H), 5.9 Hz, J2 = 30.5 Hz, 1H), 7.21–7.30 (m, 1H), 7.35 (t, J = 1.5 Hz,
7.67–7.73 (m, 1H). MS (ESI) m/z 328.2 [M − H]−.
2H), 7.63 (s, 1H). MS (ESI) m/z 555.2 [M + H]+.
(R)-2-(2,5-Dichlorobenzamido)-3-(methylthio)propanoic acid
((R)-1-((R)-2-(2,5-Dichlorobenzamido)-3-(methylthio)pro-
1
(3c). A white solid (1.7 g, 43.2%). Mp 139.1–140.7 °C. H NMR panamido)-3-methylbutyl)boronic acid (8n). To a solution of
(400 MHz, DMSO-d6) δ 2.12 (s, 3H), 2.76–2.85 (m, 1H), 7n (2.1 g, 3.9 mmol) and isobutyl boronic acid (1.9 g,
2.91–3.01 (m, 1H), 4.56 (dd, J1 = 4.9 Hz, J2 = 9.0 Hz, 1H), 7.45 19.3 mmol) dissolved in methanol (25 mL) and hexane
(t, J = 1.4 Hz, 1H), 7.54 (dd, J1 = 1.4 Hz, J2 = 7.2 Hz, 2H), 8.95 (25 mL) was added 1 N HCl (10 mL). The mixture was stirred at
(d, J = 8.1 Hz, 1H), 12.96 (s, 1H). MS (ESI) m/z 306.1 [M − H]−.
room temperature for 22 h. The methanolic phase was washed
2,5-Dichloro-N-((S)-1-cyclohexyl-2-(((R)-3-methyl-1-((3aS,4S, with hexane (3 × 25 mL), and the hexane layer was extracted
6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2] with methanol (3 × 25 mL). The combined methanolic layers
dioxaborol-2-yl)butyl)amino)-2-oxoethyl)benzamide (7i). To a were evaporated in vacuo, and the residue was dissolved in
cooled solution (−10 °C) of 3a (0.3 g, 0.9 mmol) dissolved in CH2Cl2 (20 mL). The solution was washed with brine (3 ×
anhydrous CH2Cl2 (20 mL) was added HOBt (0.2 g, 1.4 mmol). 20 mL), and the organic layer was dried over anhydrous
After 10 min, EDCI (0.3 g, 1.4 mmol) was added. Finally, 5a Na2SO4, evaporated, and purified by chromatography to give
(0.3 g, 0.9 mmol) and DIPEA (0.4 g, 0.32 mmol) were added. 0.8 g of yellow foam solid (52.4%).1H NMR (400 MHz, CDCl3)
The mixture was stirred at −10 °C for 1 h and at room tempera- δ 0.87 (s, 6H), 1.37–1.48 (m, 2H), 1.59 (dd, J1 = 6.4 Hz, J2
=
ture for 15 h. The mixture was washed with 1 N HCl, 5% 13.2 Hz, 1H), 2.18 (s, 3H), 2.87–3.02 (m, 2H), 3.02–3.11 (m,
NaHCO3, and brine, and dried over anhydrous Na2SO4. After 1H), 4.82–5.02 (m, 1H), 7.32 (t, J = 5.9 Hz, 2H), 7.35–7.41 (m,
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1H), 7.42–7.54 (m, 1H), 7.58 (s, 1H). 13C NMR (100 MHz, 3.53–3.74 (m, 4H), 4.52–4.62 (m, 1H), 7.45 (dd, J1 = 1.4 Hz, J2 =
CDCl3) δ 15.87, 22.94, 25.83, 29.60, 39.73, 50.90, 51.22, 129.09, 15.6 Hz, 1H), 7.47–7.62 (m, 3H), 8.81 (dd, J1 = 8.3 Hz, J2 = 17.6,
129.63, 131.35, 133.07, 135.41, 165.40, 171.84. MS (ESI) m/z 1H). 13C NMR (100 MHz, DMSO-d6) δ 14.87, 21.64, 24.04,
419.2 [M − H]−. HRMS (ESI) calcd for C16H23BCl2N2NaO4S 34.55, 35.62, 50.98, 51.45, 62.52, 128.54, 128.68, 128.89,
[M + Na]+, 443.0754; found, 443.0744.
130.71, 131.49, 164.80, 169.01. HRMS (ESI): calcd for
5-(Carboxymethyl)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine- C22H27BCl2N2O10 [M + Na]+ 512.1319, found 512.1315.
2-carboxamido)propanamido)butyl)-6-oxo-1,3,2-dioxaborinane-
4-carboxylic acid (8q). To a solution of citric acid (0.2 g,
Enzyme inhibition assays
0.9 mmol) dissolved in ethyl acetate (5 mL) at 74 °C, bortezo- The enzymatic activities of the proteasome were analyzed
mib (0.3 g, 0.8 mmol) dissolved in ethyl acetate (2 mL) was using fluorogenic peptides: Suc-Leu-Leu-Val-Tyr-AMC (Suc rep-
added dropwise and then cooled slowly to 60 °C. The mixture resents succinyl and AMC represents 7-amido-4-methyl-
was stirred for 3 h. The resulting slurry was cooled slowly to coumarin, obtained from Enzo) for chymotrypsin-like (CT-L)
room temperature and the mixture was stirred overnight. The activity. Four microliters of human 20S proteasome (0.2 nM)
mixture was filtered and a white solid 8q was obtained (0.3 g, were mixed with 4 μL of various concentrations of compounds.
1
71.1%). H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 6H), 1.19 (dd, After 10 min, 8 μL of fluorogenic peptides (50 μM) was added
J1 = 12.1 Hz, J2 = 19.2 Hz, 2H), 1.44 (d, J = 33.5 Hz, 1H), 2.57 (d, and incubated at 37 °C for 1 h. The fluorescence of the
J = 13.8 Hz, 2H), 2.73 (s, 1H), 2.85 (s, 1H), 3.24 (d, J = 6.6 Hz, released AMC reagents was measured using a spectrofluori-
2H), 4.03 (q, J = 7.1 Hz, 1H), 5.06 (dd, J1 = 7.4 Hz, J2 = 15.1 Hz, meter (CLARIOstar, BMG Germany) at excitation and emission
1H), 7.18–7.32 (m, 5H), 8.77 (s, 1H), 8.90 (s, 1H), 9.14 (s, 1H), wavelengths of 360/460 nm. DMSO (1%) was used as the
9.21 (s, 1H), 10.62 (s, 1H), 12.12 (s, 2H). 13C NMR (100 MHz, solvent control. An inhibition rate was calculated and the IC50
DMSO-d6) δ 20.81, 21.45, 23.50, 24.63, 36.42, 41.28, 50.41, 59.81, value was inferred compared with the fluorescence of the
76.28, 126.87, 128.40, 129.37, 143.46, 143.79, 144.03, 147.93, solvent control.29
163.33, 170.38, 170.92, 177.08, 177.98. HRMS (ESI): calcd for
Cytotoxicity assays
C25H29BN4NaO9 [M + Na]+, 563.1924; found, 563.1945.
Compounds 8r–8t were prepared from the corresponding The cytotoxic effects of the compounds were detected using
starting materials described for the synthesis of 8q.
the CellTiter 96 Aqueous One Solution Cell Proliferation assay
N-(R)-(2-((1-(6-(2-Hydroxyethyl)-1,3,6,2-dioxazaborocan-2-yl)- (Promega). Suspension cells (RPMI8226, ARH77, U266B and
3-methylbutyl)amino)-2-oxoethyl)-2,5-dichlorobenzamide (8r). MDA-MB-231) growing in the log phase were plated at 5000
White solid (0.35 g, 53.3%). 1H NMR (400 MHz, DMSO-d6) cells per well and collected by centrifugation. Twenty-four
δ 0.80 (dd, J1 = 11.6 Hz, J2 = 23.5 Hz, 6H), 1.14–1.27 (m, 2H), hours after plating, media including the test compounds were
1.44–1.58 (m, 1H), 2.79–2.93 (m, 2H), 3.03 (s, 2H), 3.16 (dd, added to each well to afford the intended final concentration.
J1 = 5.2 Hz, J2 = 11.0 Hz, 2H), 3.21 (d, J = 5.2 Hz, 1H), 3.64–3.73 After 72 h, the cells’ viability was detected using the assay pro-
(m, 4H), 3.75 (d, J = 10.5 Hz, 2H), 3.78 (s, 1H), 4.88 (t, J = tocol recommended by the manufacturers. The resulting
5.1 Hz, 1H), 6.52 (d, J = 9.8 Hz, 1H), 7.50 (d, J = 9.1 Hz, 1H), signals were quantified using a CLARIOstar microplate reader
7.53–7.61 (m, 2H), 8.97 (t, J = 6.0 Hz, 1H). 13C NMR (100 MHz, (BMG LABTECH).30
DMSO-d6) δ 21.85, 24.23, 24.28, 42.46, 43.12, 57.20, 58.47,
Animal welfare and experimental procedures were carried
128.49, 128.76, 130.82, 131.51, 131.68, 137.81, 165.37, 166.87. out in accordance with the Guide for the Care and Use of
HRMS (ESI): calcd for C20H30BCl2N3NaO5 [M + Na]+, 496.1551; Laboratory Animals (Ministry of Science and Technology of
found, 496.1548.
China, 2006), and were approved by the animal ethics commit-
N-(R)-(2-((1-(1,3,6,2-Dioxazaborocan-2-yl)-3-methylbutyl)amino)- tee of Nanjing Normal University.
2-oxoethyl)-2,5-dichlorobenzamide (8s). White solid (0.56 g,
Pharmacokinetic studies
85.4%). 1H NMR (400 MHz, DMSO-d6) δ 0.79 (d, J = 6.4 Hz,
3H), 0.82 (d, J = 6.7 Hz, 3H), 1.12–1.39 (m, 2H), 1.60 (s, 1H), Compound 8t was administered intragastrically using
a
2.65–3.06 (m, 4H), 3.14 (dd, J1 = 6.6 Hz, J2 = 10.8 Hz, 1H), feeding tube to male SD rats at a dose of 2 mg kg−1 and caudal
3.30–3.73 (m, 4H), 3.74–3.89 (m, 2H), 6.58 (s, 1H), 6.98 (d, J = intravenously at a dose of 2 mg kg−1. Orbital blood was col-
8.1 Hz, 1H), 7.54 (d, J = 10.9 Hz, 3H), 8.83 (s, 1H). 13C NMR lected at 0 min, 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h,
(100 MHz, DMSO-d6) δ 21.67, 23.97, 24.53, 42.83, 50.49, 50.92, 8 h, 12 h, 24 h and 30 h after the oral administration, and at
62.44, 128.67, 128.85, 130.75, 131.50, 131.61, 137.83, 165.25, 0 min, 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h,
167.73. HRMS (ESI): calcd for C18H26BCl2N3NaO4 [M + Na]+, 24 h after the tail intravenous administration. The blood
452.1289; found, 452.1299.
samples were centrifuged at 3500 rpm for 10 min to obtain the
N-((R)-1-(((R)-1-(1,3,6,2-Dioxazaborocan-2-yl)-3-methylbutyl) plasma fraction, and 200 μL of plasma were stored at −25 °C
amino)-3-(methylthio)-1-oxopropan-2-yl)-2,5-dichlorobenzamide until analysis. The drug concentrations in the plasma were
(8t). White solid (0.23 g, 75.4%). 1H NMR (400 MHz, DMSO-d6) quantitatively analyzed using the liquid chromatography-
δ 0.75–0.85 (m, 6H), 1.09–1.27 (m, 2H), 1.53–1.70 (m, 1H), 2.10 tandem
mass
spectrometry
method
(LC-MS/MS).
(d, J = 1.5 Hz, 3H), 2.61–2.83 (m, 4H), 2.94–3.00 (m, 1H), Pharmacokinetic parameters were determined by a non-com-
3.08–3.18 (m, 1H), 3.45 (dd, J1 = 8.1 Hz, J2 = 13.6 Hz, 1H), partmental analysis using the WinNonlin 6.3 program.
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Pharmacodynamics studies
11 S. Lonial, E. K. Waller, P. G. Richardson, S. Jagannath,
R. Z. Orlowski, C. R. Giver, D. L. Jaye, D. Francis, S. Giusti,
C. Torre, B. Barlogie, J. R. Berenson, S. Singhal,
D. P. Schenkein, D. L. Esseltine, J. Anderson, H. Xiao,
L. T. Heffner and K. C. Anderson, Blood, 2005, 106, 3777–3784.
12 X. Cai, S. Bhattacharyya, A. Plitt, R. Raibagkar,
J. N. LaBuzetta, S. M. Schleicher, N. D. Munshi and
J. P. Kleain, J. Clin. Oncol., 2014, 34, e1–e5.
Rats received one single dose of MLN9708 and 8t by ig admin-
istration (5 mg kg−1) for the indicated times and harvested. At
selected time points after the drug administration, approxi-
mately 100 μL of whole blood was collected from each rat and
processed for the 20S proteasome activity assay.
In vivo antitumor activity
13 S. Atrash, A. Tullos, S. Panozzo, M. Bhutani, F. Van Rhee,
B. Barlogie and S. Z. Usmani, Blood Cancer J., 2015, 5, e272.
14 R. D. Harvey, Clin. Pharmacol., 2014, 6, 87–96.
15 D. J. Fall, H. Stessman, S. S. Patel, Z. Sachs, B. G. Van Ness,
L. B. Baughn and M. A. Linden, J. Cancer, 2014, 5, 720–727.
16 D. Niewerth, G. Jansen, Y. G. Assaraf, S. Zweegman,
G. J. Kaspers and J. Cloos, Drug Resist. Updates, 2015, 18,
18–35.
The MDA-MB-231 cells (5 × 106) were subcutaneously injected
into BALB/c (nu/nu) mice. The mice were distributed randomly
and treated with compound 8t (1.15 mg per kg body weight,
Q2D) for 14 days. Then the mice were sacrificed and tumors
were removed and weighed. The tumors were dyed using
hematoxylin and eosin (HE) and histopathological analysis
was performed under a light microscope.
17 S. K. Kumar, J. H. Lee, J. J. Lahuerta, G. Morgan,
P. G. Richardson, J. Crowley, J. Haessler, J. Feather,
A. Hoering, P. Moreau, X. LeLeu, C. Hullin, S. K. Kleain,
P. Sonneveld, D. Siegel, J. Blade, H. Goldschmidt,
S. Jagannath, J. San Miguel, R. Orlowski, A. Palumbo,
O. SEzer and B. G. M. Durie, Leukemia, 2012, 26, 149–157.
18 E. S. Weyburne, O. M. Wilkins, Z. Sha, D. A. Williams,
A. A. Pletnev, G. Bruin, H. S. Overkleeft, A. L. Goldberg,
M. D. Cole and A. F. Kisselev, Cell Chem. Biol., 2017, 24,
218–230.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the following grants: the National
Natural Science Foundation of China (21877061), the Natural
Science Foundation of Jiangsu Province (BK20171448), the
National and Local Joint Engineering Research Center of
Biomedical Functional Materials and the Priority Academic
Program Development of Jiangsu Higher Education Institutions
(PAPD).
19 M. Rolfe, Cell Chem. Biol., 2017, 24, 125–126.
20 E. A. Rakha, M. E. EI-Sayed, A. R. Green, A. H. Lee,
J. F. Robertson and I. O. Eliis, Cancer, 2007, 109, 25–32.
21 M. Tischkowitz, J. S. Brunet, L. R. Bégin, G. H. David,
M. C. Cheang, L. A. Akslen, T. O. Nielsen and
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