Design, synthesis, and biological evaluation of bone-targeted proteasome inhibitors for multiple myeloma

Article abstract of DOI:10.1016/j.bmcl.2013.09.043

Multiple myeloma (MM) is an incurable neoplasm characterized by devastating and progressive bone destruction. Standard chemotherapeutic agents have not been effective at significantly prolonging the survival of MM patients and these agents are typically a

Full text of DOI:10.1016/j.bmcl.2013.09.043

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6455–6458
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of bone-targeted
proteasome inhibitors for multiple myeloma
a,b,
⇑
, Bindu Santhamma c, , Sudipa S. Roy a,b
Joseph K. Agyin
a
University of Texas Health Science Center at San Antonio, Biochemistry Department, 7703 Floyd Curl Drive, San Antonio, TX 78229, United States
University of Texas Health Science Center at San Antonio, Cellular and Structural Biology Department, 7703 Floyd Curl Drive, San Antonio, TX 78229, United States
Evestra Inc., 7620 N.W. Loop 410, San Antonio, TX 78227, United States
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2013
Revised 10 September 2013
Accepted 13 September 2013
Available online 21 September 2013
Multiple myeloma (MM) is an incurable neoplasm characterized by devastating and progressive bone
destruction. Standard chemotherapeutic agents have not been effective at significantly prolonging the
survival of MM patients and these agents are typically associated with often severe, dose-limiting side
effects. There is great need for methods to target the delivery of novel, effective cytotoxic agents specif-
ically to bone, where myeloma cells reside. We have synthesized and evaluated the effects of the bone-
targeted proteasome inhibitors PS-341-BP-1, PS-341-BP-2 and MG-262-BP on cell proliferation using the
mouse 5TGM1 and human RPMI 8226 cell lines in vitro. The compounds exhibit strong cytotoxicity on
MM cell lines and reduce the number of viable cells in a dose dependent manner.
Keywords:
Multiple myeloma
Proteasome inhibitor
Bone-targeted
Ó 2013 Elsevier Ltd. All rights reserved.
Bisphosphonate conjugate
Boronic acid
Several proteasome inhibitors have been identified, including
peptide boronates (Bortezomib, its bidendate analog BU-32, and
MG-262), epoxiketones (Carfilzomib, also called Kyprolis, which
has recently been approved for treatment of multiple myeloma
MM) patients by the FDA), PR047, and Epoxomicin, peptide alde-
is an urgent need for new approaches to selectively target the
delivery of effective anticancer drugs such as bortezomib specifi-
cally to bone tissue, where myeloma cells reside, to ameliorate
and/or prevent the toxic side effects arising from systemic distribu-
tion of these drugs and to make dose escalation possible in order to
improve therapeutic outcome.
1
(
hydes (MG-132 and PSI), and b-lactones (NPI-0052, also called sal-
inosporamide A and lactacystin).2–5 Several proteasome inhibitors
that are currently in clinical trials including MLN9708, CEP-18770,
Carfilzomib, PR-047, and NPI-0052, a b-lactone.6 In addition, allo-
steric chemical inhibitors that target the proteasome outside of
the active site have been developed, including chloroquine and
its analog 5-amino-8-hydroxyquinoline (5AHQ).7 The inhibitory
potency and target selectivity towards the 20S proteasome of boro-
nic acid-based inhibitors are quite remarkable (in the nM range).
This is presumably due to the fact that an empty p-orbital on a bor-
on atom is positioned to accept the oxygen lone pair of the amino
terminal threonine residue of the 20S proteasome to form a stable
Almost all patients with MM have an early excessive bone
resorption leading to lytic lesions and sometimes to hypercalce-
9,10
mia.
Interactions between myeloma cells and bone cells and
the extracellular matrix proteins within the bone microenviron-
ment underlie the progression of multiple myeloma and are med-
11
iated through cell surface receptors. These interactions trigger a
self-amplifying cascade of events that result in the secretion of
cytokines and growth factors that promote the growth and prolif-
eration of myeloma cells, increase bone resorption and enhance
1
1–13
drug resistance by inducing antiapoptotic pathways.
Bisphos-
phonates, which are degradation-resistant analogs of inorganic
pyrophosphate, exhibit exceptional affinity for bone and have tra-
ditionally been the mainstay therapy for the treatment of skeletal
8
tetrahedral intermediate. To date, Bortezomib and carfilzomib are
the only proteasome inhibitors approved for treatment of MM. De-
spite its remarkable effect in myeloma therapy, significant toxici-
ties, such as peripheral neuropathy and thrombocytopenia, have
restricted the intensity of bortezomib dosing and its clinical
efficacy has been hampered by the emergence of resistance. There
1
3
related events associated with myeloma. However, there may be
a greater role for the use of bisphosphonates than has previously
been considered. Recent reports suggest that they may act as anti-
1
4
tumor agents, able to delay disease progression and prolong sur-
1
5
16
vival in multiple myeloma, and in solid tumors such as breast
and prostate cancers.
1
7,18
These observations, together with the
⇑
Corresponding author. Tel.: +1 210 567 0796; fax: +1 210 567 3803.
E-mail address: agyinj@uthscsa.edu (J.K. Agyin).
Current address.
efficacy of proteasome inhibitors in myeloma treatment and recent
reports that suggest that proteasome inhibitors may stimulate new
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.043

6
456
J. K. Agyin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6455–6458
bone formation,19,20 strongly argue for the use of proteasome
inhibitor-bisphosphonate conjugates to target these potent drugs
selectively to bone tissue in order to reduce off-target adverse
effects and improve treatment outcomes. The important message
is that by targeting proteasome inhibitors to bone, it may be pos-
sible to accentuate the beneficial bone anabolic effects of these
O
H
N
B
HCl
.
H2N
O
O
5
O
O
O
N
O
O
B
a
N
b
N
c
N
d
H
N
OH
OCH3
OH
N
N
N
H
O
N
HO
H3CO
3
H CO
N
N
N
H CO
O
3
O
2
O
O
1
3
4
O
6
e
O
OH
O
P
H
O
O
B
HO
N
N
N
B
H
OH
N
H
OH
f
N
N
N
HO
P
O
H
N
N
O
O
H
HO
O
HO
OH
O
7
O
PS-341-BP-1
Scheme 1. Synthesis of PS-341-BP-1. Reagents and conditions: Key: (a) SeO
2 2
, H O, pyridine, 68%; (b) MeOH, HCl gas, 94%; (c) MeOH, NaOH, 73%; (d) 5, TBTU, DIEA, DMF, 60%;
(
e) LiOH, THF/MeOH/H O, 47%; (f) (i) NHS, DCC, DME; (ii) alendronate, DIEA, 2-propanol/H
2
2
O; (iii) (2-methylpropyl)boronic acid, MeOH/Hexane/2 M HCl.
OH
OH
O
OH
+
O
B
O
O
.
a
H
b
H
TFA H2N
N
N
NB
O
NB
.
N
H
O
HCl H2N
O
OH
BocHN
O
O
O
9
10
O
8
O
P
OH
OH
O
HO
P
OH
c
N
H
O
HO
OEt
O
O
O
OH
OH
H
N
N
NB
d
N
O
O
H
H
O
N
NB
N
O
H
O
N
PS-341-BP-2
11
Scheme 2. Synthesis of PS-341-BP-2. Reagents and conditions: Key: (a) TBTU, DIEA, DMF, followed by deprotection with HCl in dioxane; (b) 2-pyrazine carboxylic acid, TBTU,
DIEA, DMF, 40%; (c) ethyl 4-bromobutyrate,
K
2
CO
3
,
2 2
DMF; (d) (i) LiOH, MeOH/H O/THF; (ii) NHS, DCC, DME; (iii) alendronate, DIEA, H O/2-propanol; (iv) (2-
methylpropyl)boronic acid, MeOH/hexane/2 M HCl
O
O
H
N
O
O
O
O
N
H
N
H
B
a
+
HCl.H2N
O
H
N
O
MeO
O
O
N
N
H
B
MeO
N
O
O
O
14
O
12
13
b, c, d
O
O
O
O
H
O
O
P
H
OH
OH
O
P
e
HO
HO
P
OH
N
HO
HO
N
OH
O
N
H
N
H
B
O
N
H
N
H
B
H
H
O
N
O
N
O
P
HO
3
OH
HO
3
OH
O
O
O
O
MG-262-BP
15
2
O; (c) NHS, DCC, DMF; (d) alendronate, DIEA, H O/2-propanol;
Scheme 3. Synthesis of MG-262-BP. Reagents and conditions: Key: (a) DMAP, CH
e) (2-methylpropyl)boronic acid, MeOH/hexane/2 M HCl.
2
Cl
2
; (b) LiOH, THF/MeOH/H
2
(

J. K. Agyin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6455–6458
6457
O
O
P
OH
HO
P
OH
O
N
H
HO OH
O
O
OH
B
O
O
O
P
H
N
O
H
OH
B
O
P
H
N
HO
HO
P
N
N
HO
HO
OH
OH
OH
N
N
N
O
N
H
N
H
B
N
H
OH
H
N
N
H
OH
H
N
O
O
OH
O
HO P
HO
OH
OH
O
O
O
O
MG-262-BP
PS-341-BP-1
PS-341-BP-2
Figure 1. Chemical structures of bone-targeted proteasome inhibitors.
compounds, kill myeloma cells more effectively, and prevent or re-
duce toxic side effects associated with the current mode of
administration.
In order to evaluate the therapeutic potential of bone-targeted
proteasome inhibitors, we used the procedures outlined in
Schemes 1–3 to synthesize a series of boronated proteasome inhib-
itor-bisphosphonate conjugates: PS-341-BP-1, PS-341-BP-2 and
MG-262-BP (Fig. 1) containing the proteasome inhibitors PS-341
and MG-262 as the active components. To synthesize PS-341-BP-
2
A
Cell viability of human mulꢀple
myeloma
1
.8
1.6
.4
(RPMI 8226) cell line PS 341
1
PS 341-BP1
PS 341-BP2
MG 262
1.2
1
MG 262-BP
1
2
(Scheme 1), 2,5-dimethylpyrazine was converted to pyrazine-
,5-dicarboxylic acid (2) according to the literature procedure.
2
1
0
0
0
.8
.6
.4
Hydrogen chloride gas was bubbled through a solution of com-
pound 2 in methanol to form the di-ester 3 which was treated with
1
.0 M sodium hydroxide to afford the mono-ester 4. The key boro-
nic acid dipeptide pinanediol ester (5) intermediate, which was
2
2,23
0.2
0
synthesized in six steps using the literature procedure,
was
coupled to compound 4 to produce 6. The methyl ester 6 was
hydrolyzed to the acid 7 which was then conjugated to the bis-
phosphonate alendronate followed by pinanediol deprotection to
afford PS-341-BP-1. The synthesis of PS-341-BP-2 was achieved
using the procedure outlined in Scheme 2. The key intermediate
3
6
9
12
15
18
Concentraꢀon (nM)
pinanediol leucine boronated trifluoroacetate salt 8 was synthe-
B
sized using the published procedure,2
2,23
and was coupled to
Cell viability of mouse mluꢀple
myeloma (5TGM1) cell line PS 341
2.5
N-Boc-tyrosine followed by deprotection of the Boc group to pro-
duce compound 9. Next, compound 9 was coupled to 2-pyrazine
carboxylic acid to give 10 which was then coupled to ethyl
2
.5
1
PS 341-BP1
PS 341-BP2
MG 262
4
-bromobutanoate to yield the corresponding ether 11. After
1
0
hydrolysis of the ethyl ester, the carboxylic acid was converted
to the N-hydroxysuccinimide ester and then coupled to alendro-
nate to give the pinanediol boronate ester which was deprotected
to afford PS-341-BP-2. Next, the synthesis of MG-262-BP was
achieved using the procedure outlined in Scheme 3. 4-(Methoxy-
carbonyl)benzyl 1H-imidazole-1-carboxylate 12 was reacted with
the boronated tripeptide 13 to give the carbamate intermediate
MG 262-BP
.5
0
1
4. After hydrolysis of the methyl ester and activation of the car-
boxylic acid followed by reaction with alendronate, compound
5 was obtained. After deprotection of the boronate, MG-262-BP
3
6
9
12
15
18
Concentraꢀon (nM)
1
was obtained. Details of the experimental procedures are provided
in the Supplementary data.
Figure 2. Effect of proteasome inhibitor on myeloma cell proliferation. Proliferation
assay of RPMI8226 (A) and 5TGM1 (B) myeloma cells were treated with different
concentrations (1–18 nM) of proteasome inhibitors for 72 h. Results are mean of
three independent experiments.
The sulforhodamine B assay (SRB assay) was used to evaluate
the antiproliferative and cytotoxic effects of the bone-targeted pro-
teasome inhibitors PS-341-BP-1, PS-341-BP-2 and MG-262-BP
using the mouse 5TGM1 and human RPMI 8226 cell lines in vitro
2
4,25
from 4.89 to 9.39 nM compared with 6.78 nM for PS-341 in 5TGM1
and 10.18–13.76 nM compared with 9.50 nM for PS-341 in RPMI
as described.
RPMI 8226 and 5TGM1 cells were purchased
from the American Type Culture Collection. The cell lines were
seeded in triplicate at 3000 cells per well in 96-well plates in RPMI
containing 10% fetal bovine serum. After 24 h, the cells were trea-
ted with various drug concentrations. Colorimetric analyses were
done after 72 h of growth in the presence of drug. The IC50 repre-
sents the mean of at least three independent experiments using
triplicate points in each experiment. The results of the SRB assay
revealed that all three BP conjugates inhibited cell viability and
proliferation, after 72 h of treatment, in a dose-dependent manner
8
226 (Table 1). The choice of a suitable linker for the bisphospho-
nate is of fundamental importance to the success of these bone-
targeted pro-drug conjugates, as linkers that are too stable cannot
release the drug whereas linkers that are too labile result in prema-
ture release of the drug. To ensure ready release of the proteasome
inhibitors in the tumor microenvironment, we plan to investigate a
series of BP-drug pharmacophores containing more labile linkers
such as carbamate, carbonate, and ester moieties to determine
the best linker for targeting these drugs to bone. The use of
(Fig. 2), with a half maximal inhibitory concentration (IC50) ranging

6
458
J. K. Agyin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6455–6458
Table 1
Cell viability was measured with SRB assay reagent after compound exposure for 48 h
Cell line
TGM1
RPMI8226
PS-341 (nM)
PS341-BP-1 (nM)
PS341-BP-2 (nM)
MG-262 (nM)
MG-262 BP (nM)
5
6.78 + 0.57
9.50 + 0.48
7.59 + 0.68
10.18 + 0.87
9.39 + 0.78
11.51 + 0.92
9.18 + 0 .54
13.76 + 0.89
4.89 + 0.12
12.47 + 0.87
⁄
IC50 values were determined in each of the cell lines from a graph of at least six drug concentrations that span the entire curve of growth inhibition as means of three
independent experiments ± SD.
bone-targeted nanoparticles loaded with these drugs will be an
alternative approach to deliver them to the bone microenviron-
ment, which we are currently investigating.
In summary, we have synthesized a series of proteasome inhib-
itor-bisphosphonate conjugates that are potent inhibitors of cell
viability and proliferation in vitro in 5TGM1 and RPMI 8226 mye-
loma cell lines. This study could serve as a basis to provide prom-
ising bone-targeted drugs for effective treatment of multiple
myeloma. We plan to address this in future studies.
Jamal, N.; Messner, H.; Cloos, J.; Rose, D. R.; Navon, A.; Guns, E.; Batey, R. A.;
Kay, L. E.; Schimmer, A. D. J. Natl. Cancer Inst. 2010, 102, 1069.
Myung, J.; Kim, K. B.; Crews, C. M. Med. Res. Rev. 2001, 21, 245.
Barille, S.; Akhoundi, C.; Collette, M.; Mellerin, M. P.; Rapp, M. J.; Harousseau, J.
L.; Bataille, R.; Amiot, M. Blood 1997, 90, 1649.
8
9
.
.
1
1
1
0. Roodman, G. D. J. Cell. Biochem. 2010, 109, 283.
1. Corso, A.; Ferretti, E.; Lazzarino, M. Hematology 2005, 10, 215.
2. Raab, M. S.; Podar, K.; Breitkreutz, I.; Richardson, P. G.; Anderson, K. C. Lancet
2009, 374, 324.
3. Modi, N. D.; Lentzsch, S. Leukemia 2012, 26, 589.
4. Costa, L.; Harper, P.; Coleman, R. E.; Lipton, A. Crit. Rev. Oncol. Hematol. 2011,
1
1
7
7, S31.
1
5. Morgan, G. J.; Davies, F. E.; Gregory, W. M.; Cocks, K.; Bell, S. E.; Szubert, A. J.;
Navarro-Coy, N.; Drayson, M. T.; Owen, R. G.; Feyler, S.; Ashcroft, A. J.; Ross, F.;
Byrne, J.; Roddie, H.; Rudin, C.; Cook, G.; Jackson, G. H.; Child, J. A. Lancet 2010,
Acknowledgements
3
76, 1989.
This work was supported by Grants KO1 CA113468 and P30
CA054174 (to J.K.A.) from the National Cancer Institute, and by
Grants from the Kerr, and William & Ella Owens Research Founda-
tions (J.K.A.).
16. Gnant, M.; Mlineritsch, B.; Schippinger, W.; Luschin-Ebengreuth, G.;
Postlberger, S.; Menzel, C.; Jakesz, R.; Seifert, M.; Hubalek, M.; Bjelic-Radisic,
V.; Samonigg, H.; Tausch, C.; Eidtmann, H.; Steger, G.; Kwasny, W.; Dubsky, P.;
Fridrik, M.; Fitzal, F.; Stierer, M.; Rucklinger, E.; Greil, R.; Marth, C. N. Eng. J.
Med. 2009, 360, 679.
7. Smith, M. R.; Saad, F.; Coleman, R.; Shore, N.; Fizazi, K.; Tombal, B.; Miller, K.;
Sieber, P.; Karsh, L.; Damiao, R.; Tammela, T. L.; Egerdie, B.; Van Poppel, H.;
Chin, J.; Morote, J.; Gomez-Veiga, F.; Borkowski, T.; Ye, Z.; Kupic, A.; Dansey, R.;
Goessl, C. Lancet 2012, 379, 39.
1
Supplementary data
1
1
2
8. Coleman, R.; Gnant, M.; Morgan, G.; Clezardin, P. J. Natl. Cancer Inst. 2012, 104,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.09.
1059.
9. Pennisi, A.; Li, X.; Ling, W.; Khan, S.; Zangari, M.; Yaccoby, S. Am. J. Hematol.
2009, 84, 6.
0. Zangari, M.; Esseltine, D.; Lee, C. K.; Barlogie, B.; Elice, F.; Burns, M. J.; Kang, S.
H.; Yaccoby, S.; Najarian, K.; Richardson, P.; Sonneveld, P.; Tricot, G. Br. J.
Haematol. 2005, 131, 71.
0
43.
References and notes
2
1. Schut, W. J.; Mager, H. I. X.; Berends, W. J. R. Neth. Chem. Soc. 1961, 80, 391.
1
.
Agyin, J. K.; Santhamma, B.; Nair, H. B.; Roy, S. S.; Tekmal, R. R. Breast Cancer Res.
009, 11, R74.
Traenckner, E. B.; Wilk, S.; Baeuerle, P. A. EMBO J. 1994, 13, 5433.
Meng, L.; Mohan, R.; Kwok, B. H.; Elofsson, M.; Sin, N.; Crews, C. M. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 10403.
22. Shenvi, A. B.; Kettner, C. A.; Patent, U. S., Ed. USA, 1985, Vol. 4,499,082.
23. Adams, J.; Behnke, M.; Chen, S.; Cruickshank, A. A.; Dick, L. R.; Grenier, L.;
Klunder, J. M.; Ma, Y. T.; Plamondon, L.; Stein, R. L. Bioorg. Med. Chem. Lett. 1998,
8, 333.
24. Risinger, A. L.; Jackson, E. M.; Polin, L. A.; Helms, G. L.; LeBoeuf, D. A.; Joe, P. A.;
Hopper-Borge, E.; Luduena, R. F.; Kruh, G. D.; Mooberry, S. L. Cancer Res. 2008,
68, 8881.
25. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
2
2
3
.
.
4
5
.
.
Mc Cormack, T.; Baumeister, W.; Grenier, L.; Moomaw, C.; Plamondon, L.;
Pramanik, B.; Slaughter, C.; Soucy, F.; Stein, R.; Zuhl, F.; Dick, L. J. Biol. Chem.
1
997, 272, 26103.
Craiu, A.; Gaczynska, M.; Akopian, T.; Gramm, C. F.; Fenteany, G.; Goldberg, A.
L.; Rock, K. L. J. Biol. Chem. 1997, 272, 13437.
Dick, L. R.; Fleming, P. E. Drug Discovery Today 2010, 15, 243.
Li, X.; Wood, T. E.; Sprangers, R.; Jansen, G.; Franke, N. E.; Mao, X.; Wang, X.;
Zhang, Y.; Verbrugge, S. E.; Adomat, H.; Li, Z. H.; Trudel, S.; Chen, C.; Religa, T. L.;
6
7
.
.