Evaluating the potential of Vacuolar ATPase inhibitors as anticancer agents and multigram synthesis of the potent salicylihalamide analog saliphenylhalamide

Article abstract of DOI:10.1016/j.bmcl.2008.07.003

The natural product salicylihalamide is a potent inhibitor of the Vacuolar ATPase (V-ATPase), a potential target for antitumor chemotherapy. We generated salicylihalamide-resistant tumor cell lines typified by an overexpansion of lysosomal organelles. We also found that many tumor cell lines upregulate tissue-specific plasmalemmal V-ATPases, and hypothesize that tumors that derive their energy from glycolysis rely on these isoforms to maintain a neutral cytosolic pH. To further validate the potential of V-ATPase inhibitors as leads for cancer chemotherapy, we developed a multigram synthesis of the potent salicylihalamide analog saliphenylhalamide.

Full text of DOI:10.1016/j.bmcl.2008.07.003

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5879–5883
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluating the potential of Vacuolar ATPase inhibitors as anticancer agents and
multigram synthesis of the potent salicylihalamide analog saliphenylhalamide
Sylvain Lebreton a, Janis Jaunbergs a, Michael G. Roth a,b, Deborah A. Ferguson c, Jef K. De Brabander a,b,
*
a Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9038, USA
b Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9038, USA
c Reata Pharmaceuticals, Inc., 2801 Gateway Drive, Suite 150, Irving, TX 75063, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The natural product salicylihalamide is a potent inhibitor of the Vacuolar ATPase (V-ATPase), a potential
target for antitumor chemotherapy. We generated salicylihalamide-resistant tumor cell lines typified by
an overexpansion of lysosomal organelles. We also found that many tumor cell lines upregulate tissue-
specific plasmalemmal V-ATPases, and hypothesize that tumors that derive their energy from glycolysis
rely on these isoforms to maintain a neutral cytosolic pH. To further validate the potential of V-ATPase
inhibitors as leads for cancer chemotherapy, we developed a multigram synthesis of the potent salicyli-
halamide analog saliphenylhalamide.
Received 2 June 2008
Revised 26 June 2008
Accepted 1 July 2008
Available online 5 July 2008
Keywords:
Natural product
Total synthesis
Cancer
Ó 2008 Elsevier Ltd. All rights reserved.
Vacuolar ATPase
As products of evolution, natural products are selected for inter-
action with living systems. As such, an unbiased quest to study
their function will inevitably lead to discoveries in biology, poten-
tially with therapeutic implications.1 By accessing congeners for
mode-of-action studies, optimization of potency and pharmacoki-
netic, toxicological, and metabolic properties, synthesis takes cen-
ter stage as an enabling tool to execute a natural product-based
discovery and development program.2 Herein, we report our ef-
forts to validate inhibition of the Vacuolar ATPase (V-ATPase),
the target of salicylihalamide, as a strategy for cancer chemother-
apeutic intervention. This program led to the selection and multi-
gram synthesis of a salicylihalamide analog saliphenylhalamide (2,
SaliPhe).
guishes them from previously identified V-ATPase inhibitors.6 Our
biochemical studies utilizing a reconstituted, fully purified bovine
brain V-ATPase confirmed this activity and demonstrated that Sal-
iA binds irreversibly to the trans-membranous proton-transloca-
ting domain via N-acyl iminium chemistry.7 Structure–activity
relationship studies revealed that a macrocyclic benzolactone with
a hydrophobic N-acyl enamine side-chain is essential for potent V-
ATPase inhibition and cytotoxic activity, with SaliPhe (2) equipo-
tent to SaliA.4a,b,8
Ph
HN
HN
The marine metabolite salicylihalamide A (1),3 the first member
of a family of marine and terrestrial metabolites characterized by a
signature N-acyl-enamine appended macrocyclic salicylate, has
elicited a great deal of interest from the synthetic community4—
certainly due in part because of their growth-inhibitory activities
against cultured human tumor cells and oncogene-transformed
cell lines through mechanisms distinct from standard clinical anti-
tumor agents.5 The cellular target of SaliA remained elusive until
after our first total synthesis,3b when Boyd and coworkers reported
that SaliA and other related benzolactone enamides inhibit V-ATP-
ase activity in membrane preparations of mammalian cells, but not
V-ATPases from yeast and other fungi—an observation that distin-
O
O
OH
O
OH O
OH
OH
O
O
Salicylihalamide A
Saliphenylhalamide
(1, SaliA)
(2, SaliPhe)
Although V-ATPases have been extensively explored as a therapeu-
tic target to treat osteoporosis, many lines of evidence support the
notion that they represent a potential target for treating solid tu-
mors that grow in a hypoxic and acidic micro-environment.9 In-
creased V-ATPase activity is postulated to be required for the
efficient and rapid removal of protons generated by increased rates
* Corresponding author. Tel.: +1 214 648 7808; fax: +1 214 648 0320.
E-mail address: jef.debrabander@utsouthwestern.edu (J.K. De Brabander).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.003

5880
S. Lebreton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5879–5883
of glycolysis.9b,c Maintaining a slightly basic cytosolic pH protects
the cytoplasm from acidosis and prevents apoptosis, and acidificat-
ion of the extracellular environment promotes invasion,10 metasta-
sis, immune suppression,11 and resistance to radiation and
chemotherapy.9 Proper V-ATPase function is also crucial for the exe-
cution of the autophagic pathway, which has been implicated as a
protective mechanism in cancer.12 To demonstrate that inhibition
of V-ATPase activity is related to the toxicity induced by salicyliha-
lamide, we have created various drug-resistant cell lines by cultur-
ing human melanoma cells (SK-MEL-5) in increasing concentrations
of SaliA. A cell line resistant to 100 nM of SaliA (SR100) possessed a
phenotype distinguished by an increased number of acidic lyso-
somal organelles (Fig. 1A). Western blot analysis indicated that V-
ATPase subunits and lysosomal membrane proteins are strongly
upregulated in this resistant cell line (Supplementary data Fig.
S1). An independent derived cell line resistant to 40 nM of SaliA
(SR40) also displays an increased number of larger lysosomes as
compared to drug-sensitive SK-MEL-5 cells as shown by staining
with antibodies specific for the lysosomal marker proteins CD63
and Lamp2 (Fig 1B). Our working hypothesis is that the more malig-
nant tumors rely on V-ATPase activity to deal with increased acid-
load from glycolysis,13 and exploit otherwise tissue-specific iso-
forms found on the cell surface of acid-extruding cells (osteoclasts,
kidney intercalated cells, and testis acrosomes) to maintain their
cytosolic pH. In support of this mechanism, we have found that
the majority of a set of 28 human tumor cell lines of different ori-
gins over-express such plasmalemmal isoforms as determined by
RT-PCR. As shown in Figure 2, the plasmalemmal V-ATPase E2-sub-
unit (ATP6V1E2) is highly expressed in cancer cell lines, but not in
the normal fibroblast cell lines IMR-90 and BJ. In normal human tis-
sues, expression of this subunit is highly enriched in the testis
where it functions to acidify the acrosome.14
An analog of saliA, saliphenylhalamide (SaliPhe, 2) was selected
for further preclinical evaluation based on (1) in vitro cytotoxicity
and V-ATPase inhibitory activity comparable to SaliA4b; (2) stabil-
ity and ease of synthesis;15 and (3) its differential effects on normal
and tumorigenic human mammary epithelial cells.16 As shown in
Table 1, SaliPhe is a potent antiproliferative agent with activity
Figure 1. (A) Parental SK-MEL-5 cells cultured in the absence of drug and SaliA-resistant cells growing in 100 nM SaliA (SR100) were stained with the pH-sensitive dye
Lysotracker Green (Invitrogen) according to the manufacturer’s recommendations. The dye accumulates in acidic organelles, which are few and small in the parental SK-MEL-
5 cells (top left panel), and numerous and swollen in SR100 cells (bottom left panel). (B) Parental SK-MEL-5 cells and a drug-resistant line grown continually in 40 nM SaliA
(SR40) were stained with antibodies to the lysosomal proteins CD63 (top panels) or Lamp2 (bottom panels) followed by labeling with a secondary antibody conjugated to an
Alexa 488 fluorescent dye. The drug-resistant line shows increased numbers of larger lysosomal vesicles (right panels) versus the parental SK-MEL-5 cell line (left panels).
Figure 2. ATP6V1E2 gene expression is highly enriched in the human testis (top panel) and is also highly expressed in many human tumor cell lines (middle and bottom
panels) as determined by RT-PCR. S9 ribosomal RNA served as a control for amplification.

S. Lebreton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5879–5883
5881
Table 1
R
O
Growth-inhibitory activity of SaliPhe (2) against a panel of human tumor cell lines
HN
PGO
a
Cell line
IC50 (nM)
Cell line
IC50 (nM)
OH
OPG
O
O
Hep-G2b
SK-HEP-1b
HCT-116c
HCT-15c
HT-29c
79.2 8.9
40.7 5.8
69.3 4.9
100.1 10.8
89.9 10.6
424.6 73.2
105.9 9.6
98.5 17.2
153.9 14.9
48.8 5.7
MG-63e
448.6 33.4
75.9 11.3
123.6 36.9
155.5 22.8
160.0 3.3
179.8 57.4
52.9 6.9
77.2 13.1
145.6 2.1
76.4 32.2
OH
OPG
O
O
AsPC-1f
Panc-1f
MCF-7g
MCF-7/Doxg
MDA-MB-231g
NCI-H460h
NCI-H1299h
A549h
1: R = Z,Z-(CH=CH)2Et (SaliA)
2: R = C2Ph (SaliPhe)
I
SW-480c
SK-MEL-5d
SK-MEL-28d
A2058d
OPG
OPG
OPG
Mitsunobu
RCM
PG = Protecting
Group
CO2H
HOSe
SK-MES-1h
HO
II
a
IC50 values are average standard deviation of three experiments.
b
c
d
e
f
III
Cancer type (CT) = liver.
CT = colon.
CT = melanoma.
CT = osteosarcoma.
CT = pancreas.
CT = breast.
Scheme 1. Synthesis of salicylihalamides: synthetic strategy.
g
h
of 8%.4b We have now developed a more convergent route to the
more challenging fragment III, and optimized the final steps of
our total synthesis. These efforts culminated in a scalable total
synthesis delivering 2.54 g of SaliPhe in 15–17 steps (longest lin-
ear sequence) with an improved overall yield of 9.5% (Scheme
2).
CT = lung.
against a wide variety of human tumor cell lines. There was no dif-
ference between the concentrations of SaliPhe required to inhibit
the growth of MCF-7 or MCF-7/Dox cells, the latter of which are
resistant to doxorubicin and other chemotherapeutic agents that
are substrates for P-glycoprotein (Supplementary data Table S1).
Remarkably, we observed a correlation between the resistance of
selected human tumor cell lines to radiation and their correspond-
ing sensitivity to pharmacological treatment with SaliPhe. That is,
cells that are more resistant to radiation are more sensitive to Sal-
iPhe (Fig. 3). These results are consistent with the hypothesis that
cells with higher V-ATPase activity are radioresistant.12a Further-
more, SaliPhe is also able to sensitize tumor cells to chemother-
apy17 and radiation (Supplementary data Fig. S2)
In order to further evaluate these promising anticancer activ-
ities in appropriate animal models and perform rigorous preclin-
ical toxicology and pharmacokinetic studies, we needed to
develop a scalable synthesis of SaliPhe. Our published route to
salicylihalamide and analogs was based on the assembly of the
12-membered benzolactone I via a Mitsunobu esterification of
alcohol III with ortho-substituted salicylic acid derivative II, fol-
lowed by an E-selective ring-closing olefin metathesis (Scheme
1). After installation of the side-chain and final deprotection, Sal-
iPhe (2) was obtained in 23 steps from commercially available
starting materials (longest linear sequence) with an overall yield
The new synthesis of fragment III (alcohol 9 in Scheme 2) is
based on a convergent aldol reaction between methyl ketone 4
and homochiral
a-methyl-substituted aldehyde 6. Ketone 4 and
aldehyde 6 are available in 2 steps each from commercially avail-
able 1,3-butanediol and known (ꢀ)-pseudoephedrine-derived
amide 5.18 Addition of the dicyclohexylboronate ester derived from
ketone 4 to homochiral aldehyde 6 delivered aldol product 7 in
high yield (93%, 10.8 g) but low selectivity for the desired anti-Fel-
kin diastereomer (57:43).19 The use of homochiral diisopinocamp-
heyl enolboranes did not improve the selectivity and moreover
resulted in lower yields due to competing reduction of the b-hy-
droxy ketone product 7 to the corresponding diol.20 The mixture
of b-hydroxyketones 7 was silylated (? 8) followed by syn-selec-
tive ketone reduction to alcohol 9 and corresponding 3S,5S-diaste-
reomer (86% yield, 17.1 g). Mitsunobu esterification21 of the
diastereomeric alcohol mixture 9 with ortho-allyl-substituted sali-
cylic acid derivative 1022 was followed by a ring-closing olefin
metathesis.23,24 At this point, methanolysis of the crude benzolac-
tone mixture (11 and 110) enabled a facile chromatographic re-
moval of all unwanted stereoisomers (120) and yielded 5.3 g of
the desired lactone 12 as a single pure stereoisomer (25% yield
from stereoisomer mixture 9).
The final steps of the synthesis, the introduction of the essential
N-acyl enamide side-chain, follow along the lines of our published
total synthesis.4b Silylation of the phenol, oxidative deprotection of
the p-methoxybenzyl ether, and Dess–Martin periodinane25 oxida-
tion to the aldehyde set the stage for a highly Z-selective Horner–
Wadsworth–Emmons homologation to afford a,b-unsaturated allyl
ester 13 in 80% yield (4.8 g) for the 4 step sequence. Palladium(0)-
catalyzed de-allylation (? 14) and acylazide formation performed
very reproducibly on large scale, delivering 4.1 g of Curtius rear-
rangement precursor 15 in 87% yield for the 2 step sequence. Addi-
tion of the isocyanate derived from refluxing acylazide 15 in
benzene to a ꢀ78 °C solution of lithium phenylacetylide afforded
4.04 g of bis-silyl protected SaliPhe 16. Final deprotection with a
buffered solution of HF pyridine completed the synthesis of
2.54 g of SaliPhe 2.
Figure 3. The indicated cancer cell lines were treated with a range of doses of
SaliPhe or radiation and the dose of radiation (Gy) or the concentration of SaliPhe
(nM) required to inhibit growth by 50% was calculated. The radiation IC50 values are
an average of five experiments and the SaliPhe IC50 values are an average of three
experiments (Table 1). Error bars represent the standard deviation of the radiation
IC50 values. The SD for the SaliPhe IC50 values can be found in Table 1.
In conclusion, we have demonstrated that inhibition of Vacuolar
ATPase activity is responsible for the potent antiproliferative activ-
ity of the natural product salicylihalamide. We have further pro-
vided data that indicate the potential for V-ATPase inhibitors as
cancer chemotherapeutic leads. A potent analog of salicylihala-

5882
S. Lebreton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5879–5883
a) NaH, PMBCl
.
b) SO3 py
OH
Me
OPMB
Me
OPMB
OR
OPMB
3R
5R
HO
O
e) Cy2BCl
f) TBSCl
65%
O
g) Bu3SnH,
Me2AlCl
HO
includes 3S,5S isomer
3
4
6
7.9 g
5R
OTBS
c) LiNH2 BH3
d) Swern ox.
88%
86%
O
Me
Me
Me
17.1 g
OAc
57:43 ratio
>10:1 syn
Ph
CO2H
10.8 g
19.8 g
7 R = H
N
O
9
8 R = TBS
90%
Me Me OH
Me
includes 5S isomer
5
5.6 g
h) Mitsunobu
i) RCM
j) K2CO3, MeOH
Ph
10
15.5 g
HN
COX
O
k) TBSCl
l) DDQ
OPMB
OPMB
OR
O
TBSO
O
m) DMP
q) Δ;
LiC CPh
OR
O
OR
O
OR
Me
OTBS
Me
n) HWE
O
O
OTBS
Me
OTBS
Me
(diastereomers
and E/Z-isomers)
O
O
80%
.
r) HF py
13 X = Oallyl 4.8 g
o) Pd0
p) (PhO)2PN3
85%
11 R = Ac
12 R = H
11'
12'
4.04g
16 R = TBS
2 R = H
14 X = OH 4.3 g; 97%
5.3 g; 25%
2.54 g
15 X = N3
4.1 g; 90%
Scheme 2. Reagents and conditions: (a) NaH, 4-MeOBnCl, THF (76%); (b) SO3 pyridine, DMSO, NEt3, CH2Cl2 (85%); (c) LiNH2BH3, THF (99%); (d) (COCl)2, DMSO, NEt3, CH2Cl2
(91%); (e) 4, Cy2BCl, NEt3, Et2O, 0 °C ? ꢀ78 °C, add 6; MeOH, pH 7 buffer, H2O2 (93%); (f) TBSCl, imidazole, cat. DMAP, DMF (95%); (g) Me2AlCl, Bu3SnH, CH2Cl2 (86%); (h) DIAD,
Ph3P, THF (82%); (i) 9 mol% PhCHRu(PCy3)2(Cl)2, CH2Cl2; (j) K2CO3, MeOH (30% for 2 steps); (k) TBSCl, imidazole, cat. DMAP, DMF (95%); (l) DDQ, CH2Cl2/H2O (18:1) (92%); (m)
Dess–Martin periodinane, CH2Cl2 (95%); (n) (EtO)2P(O)CH2CO2CH2CHCH2, NaH, THF, 0 °C ? rt (96%); (o) 10 mol% Pd(PPh3)3, morpholine, THF (97%); (p) (PhO)2PN3, NEt3,
benzene (90%); (q) 15, benzene, reflux; then concentrate, dissolve in THF and add to LiCCPh in THF, ꢀ78 °C (90%); (r) HF pyridine, pyridine, THF, rt.
8. Lebreton, S.; Xie, X.-S.; Ferguson, D.; De Brabander, J. K. Tetrahedron 2004, 60,
9635.
and was prepared in multigram quantities via total synthesis.
9. Reviews: (a) Farina, C.; Gagliardi, S. Drug Discov. Today 1999, 4, 163; (b)
mide A, saliphenylhalamide, was selected for further evaluation
These studies highlight the power of synthetic chemistry as an en-
abling tool for implementing a natural product-based discovery
and development program.
Sennoune, S. R.; Luo, D.; Martinez-Zaguilan, R. Cell Biochem. Biophys. 2004, 40,
185; (c) Fais, S.; De Milito, A.; You, H.; Qin, W. Cancer Res. 2007, 67, 10627; (d)
Forgac, M. Nat. Rev. Mol. Cell Biol. 2007, 8, 917.
10. For an in vitro and in vivo study of the antimetastatic potential of a small-
molecule V-ATPase inhibitor, see: Supina, R.; Petrangolini, G.; Pratesi, G.;
Tortoreto, M.; Favini, E.; Dal Bo, L.; Farina, C.; Zunino, F. J. Pharmacol. Exp. Ther.
2008, 324, 15.
Acknowledgments
11. Lardner, A. J. Leukoc. Biol. 2001, 69, 522.
We thank the National Institutes of Health (CA 90349 and CA
95471), the Robert A. Welch Foundation, and Merck Research Lab-
oratories for financial support.
12. (a) Paglin, S.; Hollister, T.; Delohery, T.; Hackett, N.; McMahill, M.; Sphicas, E.;
Domingo, D.; Yahalom, J. Cancer Res. 2001, 61, 439; (b) Kroemer, G.; Jäättelä, M.
Nat. Rev. Cancer 2005, 5, 886; (c) Kondo, Y.; Kanzawa, T.; Sawaya, R.; Kondo, S.
Nat. Rev. Cancer 2005, 5, 726; (d) Mathew, R.; Karantza-Wadsworth, V.; White,
E. Nat. Rev. Cancer 2007, 7, 961.
13. Su, Y.; Zhou, A.; Al-Lamki, R. S.; Karet, F. E. J. Biol. Chem. 2003, 278, 20013.
14. Imai-Senga, Y.; Sun-Wada, G. H.; Wada, Y.; Futai, M. Gene 2002, 289, 7.
15. The double bond geometry of the dienamide side-chain of SaliA is hard to
control and maintain,4b and SaliA does suffer from decomposition upon storage
(neat or DMSO solutions).
Appendix Supplementary. data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.003.
16. At 100 nM concentration, SaliPhe inhibited the growth of the tumorigenic
human mammary epithelial cell line HME50-T (95% growth inhibition)
without affecting the growth of the parent (isogenic) human mammary
epithelial cell line HME50, whereas SaliA indiscriminatory inhibited the
growth of both cell lines (75% and 60%, respectively, at 100 nM). We thank
G. Dikmen and G. Gellert in the laboratory of J. Shay (Department of Cell
Biology, UT Southwestern Medical Center at Dallas) for performing these
experiments. For a reference on the generation of the tumorigenic HME50-T
cell line from the isogenic HME50 cell line see: Herbert, B.-S.; Gellert, G. C.;
Hochreiter, A.; Pongracz, K.; Wright, W. E.; Zielinska, D.; Chin, A. C.; Harley, C.
B.; Shay, J. W.; Gryaznov, S. M. Oncogene 2005, 24, 5262.
17. In a genome-wide synthetic lethal RNAi screen, V-ATPase was identified as a
target that specifically reduces cell-viability of a human non-small-cell lung
cancer line (NCI-H1155) in the presence of otherwise sublethal concentrations
of paclitaxel. Exposure to SaliPhe, paclitaxel, or a combination of these agents
revealed a significant collaborative impact on cell viability in the same cancer
cell line, see: Whitehurst, A. W.; Bodemann, B. O.; Cardenas, J.; Ferguson, D.;
Girard, L.; Peyton, M.; Minna, J. D.; Michnoff, C.; Hao, W.; Roth, M. G.; Xie, X.-J.;
White, M. A. Nature 2007, 446, 815.
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