Modified norcantharidins: Synthesis, protein phosphatases 1 and 2A inhibition, and anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2004.01.093

Fourteen modified norcantharidin analogues have been synthesised and screened for their ability to inhibit the serine/threonine protein phosphatases 1 and 2A. The most potent compounds found were 10 (PP1 IC₅₀= 13±5 μM; PP2A IC₅₀=7±3 μM) and 16 (PP1 IC ₅₀=18±8 μM; PP2A IC₅₀=3.2±0.4 μM). Overall, only analogues possessing at least one acidic residue at the former anhydride warhead displayed any PP1 or PP2A inhibitory action. The ability of these analogues to inhibit PP1 and PP2A correlates well with their observed anti-cancer activity against a panel of five cancer cell lines: A2780 (human ovarian carcinoma), G401 (human kidney carcinoma), HT29 (human colorectal carcinoma), H460 (human lung carcinoma) and L1210 (murine leukemia).

Full text of DOI:10.1016/j.bmcl.2004.01.093

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1969–1973
Modified norcantharidins:
synthesis, protein phosphatases 1 and 2A inhibition,
and anticancer activity
Matthew E. Hart,a A. Richard Chamberlin,a,* Cecilia Walkom,b Jennette A. Sakoffc
and Adam McCluskeyb,*
aDepartment of Chemistry, University of California at Irvine, Irvine, CA 92697, USA
bChemistry Building, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
cDepartment of Medical Oncology, Newcastle Mater Misericordiae Hospital, NSW, 2298, Australia
Received 23 July 2003; accepted 22 January 2004
Abstract—Fourteen modified norcantharidin analogues have been synthesised and screened for their ability to inhibit the serine/
threonine protein phosphatases 1 and 2A. The most potent compounds found were 10 (PP1 IC50=13Æ5 mM; PP2A IC50=7Æ3
mM) and 16 (PP1 IC50=18Æ8 mM; PP2A IC50=3.2Æ0.4 mM). Overall, only analogues possessing at least one acidic residue at the
former anhydride warhead displayed any PP1 or PP2A inhibitory action. The ability of these analogues to inhibit PP1 and PP2A
correlates well with their observed anti-cancer activity against a panel of five cancer cell lines: A2780 (human ovarian carcinoma),
G401 (human kidney carcinoma), HT29 (human colorectal carcinoma), H460 (human lung carcinoma) and L1210 (murine
leukemia).
# 2004 Elsevier Ltd. All rights reserved.
The concept that protein kinases are the ‘on-switch’
while protein phosphatases are the ‘off-switch’ for sig-
naling mechanisms is a naıve understanding of the
action of both enzyme families. There are three major
classes of protein phosphatases: tyrosine-specific, serine/
threonine-specific, and dual-specificity phosphatases.
However, we have focused on the serine-threonine
family because of their ability to influence cell cycle
control and growth. Traditionally, the serine-threonine,
family has been classified into four subtypes based on
their biological characteristics, sensitivities to specific
inhibitors, and substrate specificity (PP1, PP2A, PP2B,
and PP2C). Phosphorylation of structural and reg-
ulatory proteins is a major cell cycle control mechanism.
Cell cycle abnormalities characterise malignancy. An
intricate phosphorylation network involving interplay
between kinases and phosphatases regulates the cell
cycle.1À3
and drosophila.1,4 Loss of function mutations in both
PP1 and PP2A genes lead to a variety of defects in
mitosis. In yeast PP1 mutants are unable to complete
anaphase successfully, and are unable to instigate
chromosome segregation, while PP1 over expression is
lethal. In drosophila, PP1 mutants die at the larval-
pupal boundary as a result of defective spindle organi-
sation, abnormal sister chromatid segregation, hyper-
ploidy and excessive chromosome condensation, as well
as a delay in progression through mitosis. In yeast
PP2A deficient mutants are not viable, however,
mutants lacking one of the PP2A subunits display
defects in cell septation and separation and the cells
become multinucleated, while in drosophila it leads to
abnormal anaphase resolution.
PP1 and PP2A exert their effects by modulating the
activity of cyclin dependent kinases (cdk) and the reti-
noblastoma protein (pRb).5,6 The activation of cdk/
cyclin complexes requires the phosphorylation of a
conserved threonine residue, as well as the removal of
inhibitory phosphorylations. PP2A inhibits the activa-
tion of cdk/cyclin complexes by directly depho-
sphorylating cdks or indirectly by influencing upstream
The role of protein phosphatases in the cell cycle is illu-
strated by the effect of gene mutations observed in yeast
* Corresponding authors. Fax: +61-249-21-5472; e-mail: adam.
mccluskey@newcastle.edu.au
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.093

1970
M. E. Hart et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1969–1973
(CAK, Wee1, Myt) and downstream kinases/phospha-
tases (Cdc25). The pRb protein controls the movement
of cells into the S-phase of the cell cycle and is regulated
by PP1 and PP2A. Hyperphosphorylation of pRb
releases the transcription factor E2F stimulating entry
into the DNA synthesis phase of the cell cycle. PP1 and
PP2A inhibit this entry by maintaining pRb in a
dephosphorylated state. Other cell cycle events controlled
by PP1 and PP2A include chromosome condensation,
nuclear membrane disintegration, reorganisation of
cytoplasmic microtubules, spindle formation, chromatid
separation, nuclear membrane reassembly and cytokin-
esis. Abnormalities in these stages together with abnor-
mal cell cycle movement and checkpoint abrogation
have been described for various phosphatase inhibitors
including cantharidin (blister beetles detailed below),
fostriecin (streptomyces pulveraceus), okadaic acid
(dinoflagellates), calyculin A (marine sponge Discodermia
calyx), microcystin-LR (blue-green algae), and tauto-
mycin (streptomyces spiroverticillatus).7À9 In particular,
fostriecin and cantharidin have been shown to force
cells prematurely through the cell cycle and into mitosis
with multiple aberrant mitotic spindles subsequently
inducing apoptotic cell death.10,11
anhydride ring opening (as cantharidin binds as can-
tharidic acid); anhydride ring opening and mono-ester-
ification. Modification of the bicyclo-skeleton is
detrimental to PP1 and PP2A inhibition. Our more
recent efforts have involved the synthesis of anhydride
modified norcantharidin analogues: both ring opened
ester analogues and the cantharimides, both of which
maintained good levels of PP1 and PP2A inhibition. As
a result we postulated that the inclusion of a single car-
boxylate and an ability to access the acidic groove of
PP1 and PP2A were beneficial to inhibition. Accord-
ingly our minimalist pharmacophore explores the effects
of the amide NH of the cantharimides, and the single
carboxylate associated with our ring opened analogues
was also incorporated whilst maintaining other key fea-
tures known to be crucial for activity. We thus devel-
oped a new series of cantharidin analogues and explored
both their PP1 and PP2A inhibition and anticancer
activities.
Each of the bicyclic norcantharidin analogues was pre-
pared by minor modification of the published Diels–
Alder conditions reported for the synthesis of 3,17
followed in some cases by catalytic hydrogenation of
the resultant 5,6-dehydro adducts, and/or opening of the
respective anhydride rings with amine or alcohol
nucleophiles.18 For example, the prototype compound,
5,6-dehydronorcantharidin, was prepared on a 25 g
scale by the addition of furan (0.393 mol) to a stirred
suspension of maleic anhydride (0.159 mol) in 100 mL
of ethyl ether at ambient temperature; after 3 days the
resultant white precipitate was collected by vacuum fil-
tration, triturated briefly with more ether, and dried
under vacuum to give 5,6-dehydronorcantharidin in
96% yield. All other cycloadditions were carried out
similarly, with reaction times and yields varying
depending on the furan substituents.18 As with this
representative example, the respective cycloadducts
generally precipitated during the reaction, giving pre-
dominantly or exclusively the exo isomer as the isolated
product. For cases in which small amounts of endo iso-
mer or other impurities were detected in the proton
NMR of the isolated products, one recrystallisation
from ethyl ether provided pure material for testing.
Catalytic hydrogenations of the 5,6-alkene bonds were
carried out as previously described for similar analo-
gues,17 and the half-acid derivatives 4 and 9–16 were
obtained by treating the corresponding anhydrides with
the appropriate free amine or alcohol.18
Norcantharidin, the demethylated analogue of canthar-
idin is also a PP2A and PP1 inhibitor and has been used
in the treatment of primary hepatoma and upper gas-
trointestinal carcinomas, and it does not display
the nephrotoxicity of cantharidin. We have shown the
growth inhibition of norcantharidin (GI50 13–47 mM) in
the cancer cell lines listed in Table 2 to be slightly less
than that of cantharidin.12 Norcantharidin increased the
mean survival time of 285 reported cases with primary
hepatoma from 4.7–11.1 months, and the 1 year survi-
val rate from 17–30%, as compared to 102 patients
treated with conventional chemotherapy (5FU, hydroxy-
camptotherine, vincristine, thiophosphoramide and
mitomycin).13 As with cantharidin, norcantharidin not
only failed to induce myelosuppression but also induced
haemopoiesis via bone marrow stimulation.14 This in
vivo response was transient, lasting one week with the
white blood count returning to normal following
chronic administration. Interestingly, in mice, nor-
cantharidin has been shown to block the leukopenia
caused by cyclophosphamide, therefore, such an agent
may antagonise myelosuppression induced by other
chemotherapeutic agents.
There has been intense interest in developing potent and
selective inhibitors of PP1 and PP2A in recent years.
These attempts have included numerous analogues of
cantharidin (so far with little success)15 and the more
complex toxins such as the Microcystin analogues,
which resulted in moderate PP1 selectivity.16 We too,
have had an ongoing interest in cantharidin analogues,
an interest that was further heightened with the dis-
covery of fostriecin as a selective and potent PP2A
inhibitor with powerful anticancer activity. We and
previous researchers have shown that cantharidin
brooks little in the way of modification whilst retaining
activity against PP1 and PP2A.15 Permissible modifi-
cations include removal of the methyl substituents;
The norcantharidin analogues, 3–16, were screened for
their ability to inhibit PP1 and PP2A.12,19 Cantharidin
(1) and norcantharidin (2) were included as internal
standards. The results of the phosphatase inhibition
study are shown in Table 1. We note that analogues 4–8
were inactive against either both PP1 and PP2A. This
finding is in keeping with previous reports in this area
and our more recent development of the cantharimides,
which suggests that at least one free carboxylate is
required in the absence of the anhydride group.15e For
example, analogue 5 (–COOCH3) IC50s>100 mM, but
14 (–COOH) displayed PP1 IC50=49mM and PP2A
IC50=9.2 mM, a minimum of a 10-fold increase in

M. E. Hart et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1969–1973
Table 1. Inhibition of protein phosphatases 1 and 2A by compounds 1–16a
1971
Compd
PP1 Inhibition
PP2A Inhibition
Compd
PP1 Inhibition
PP2A Inhibition
IC50 (mM)
IC50 (mM)
0.43Æ0.10
0.57Æ0.18
0.6Æ0.12
16Æ2
13Æ5
7.7Æ1.3
7.0Æ3.0
0.96Æ0.33
25Æ6
13Æ3
60Æ23
76Æ24
29Æ11
49Æ9
>100
12Æ2
>100
>100
21Æ6
>100
>100
>100
>100
>100
9.2Æ2.9
9.2Æ1.0
>100
>100
>100
>100
18Æ8
3.2Æ0.4
a Average of three experiments in duplicate.
potency. Closer scrutiny revealed that this may be an
oversimplification of the enzyme requirements with 4
being inactive whilst displaying a free carboxylate. The
SAR data presented herein also suggests amide NH is
required. In our previous studies the free amine of the
cantharimides fulfils this requirement.15e Of greater
interest were analogues 9–14 and 16, which displayed
stronger PP1 and PP2A inhibition; note that in each of
these analogues there is a minimum of one free carbox-
ylate. The inhibition in this group of analogues varied,
in the case of PP1, from 13–76 mM; and for PP2A from
3.2–21 mM. The lead compounds cantharidin (0.43 and
0.6 mM) and norcantharidin (0.57 and 0.96 mM) were
somewhat more potent against PP1 and PP2A, respec-
tively. Analogue 15 appears to be an anomaly, display-
ing neither PP1 nor PP2A inhibition, whist the dimethyl
analogue, 16, showed quite potent PP1 (18 mM) and
PP2A (3.2 mM) inhibition (Pombo-Villar has previously
observed that the dimethyl analogue is a 0.18 mM PP2A
inhibitor15b). Taken together these data support a mini-
mum hydrophobicity requirement for the amide in
anhydride modified cantharidin analogues (greater than
methyl).
Introduction of a second carboxylate, in the amide sub-
stituent, resulted in a modest improvement in potency; 9
PP1 IC50=16 mM, PP2A IC50=7.7 mM, 11 PP1
IC50=60 mM, PP2A IC50 12 mM) versus 10 PP1 IC50=
13 mM, PP2A IC50=7 mM. The presence of the 5,6-
double bond appeared to be detrimental to inhibition,
more so with PP1 than PP2A, for example 12
PP1 IC50=76 mM, PP2A IC50=21 mM versus 13 PP1
IC50=29 mM, PP2A IC50=9 mM, and 9 PP1 IC50=16
mM, PP2A IC50=7.7 mM versus 11 PP1 IC50=60 mM,
PP2A IC50=12 mM. This may be a result of the greater
instability associated with the unsaturated species (a
greater propensity to undergo a retro-Diels–Alder).
Over all aromatic rings were well tolerated.

1972
M. E. Hart et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1969–1973
Table 2. Growth inhibition (GI50, mM) of various tumour cell lines
after 72 h continuous exposure to test compounds 1–16
Acknowledgements
We are grateful for financial support from the National
Institutes of Health (GM57550) and The Margaret
Mitchell Fund.
Compd
Tumour cell lineb
A2780
G401
HT29
GI50 mMa
H460
L1210
1
2
3
4
5
6
7
8
10Æ2
3.5Æ0.3
35Æ2.3
53Æ7.4
6.4Æ0.7
33Æ7
4.3Æ0.9
50Æ4
15Æ2
39Æ5
13Æ0.3
49Æ5.8
References and notes
30Æ8.0
57Æ7.7
43Æ1.5
81Æ1.0 >100
98Æ2.5 >100
85Æ5.0 >100
95Æ5.0 >100
98Æ2.5 >100
90Æ0.1 >100
82Æ10 >100
97Æ3.3 >100
83Æ8.8 >100
80Æ5.8 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1. McCluskey, A.; Sakoff, J. A. Mini Rev. Med. Chem. 2001,
1, 43.
2. Sakoff, J. A.; Ackland, S. P.; Baldwin, M. L.; Keane,
M. A.; McCluskey, A. Invest. New Drugs 2002, 21, 1.
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Chem. 2002, 45, 1151.
4. Wera, S.; Hemmings, B. A. Biochem. J. 1995, 311, 17.
5. Stein, G. S.; Baserga, R.; Giordano, A.; Denhardt, D. T.
The Molecular Basis of Cell Cycle and Growth Control;
Wiley-Liss: NY, 1999.
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
10
11
12
13
14
15
16
87Æ7.3
90Æ5.8 >100
88Æ7.3 >100
89Æ0.8 >100
6. Durfee, T.; Becherer, K.; Chen, L.; Yeh, S. H.; Yang, Y.;
Kilburn, A. E.; Lee, W. H.; Elledge, S. J. Genes Dev. 1993,
7, 555.
7. Lazzereschi, D.; Coppa, A.; Mincione, G.; Lavitrano, M.;
Fragomele, F.; Colletta, G. Exp. Cell Res. 1997, 234, 425.
8. Yamashita, K.; Yasuda, H.; Pines, J.; Yasumoto, K.;
Nishitani, H.; Ohtsubo, M.; Hunter, T.; Sugimura, T.;
Nishimoto, T. EMBO J. 1990, 9, 4331.
40Æ10
25Æ1.5
49Æ15 41Æ9.2
48Æ1.5
a GI50 is the concentration that induces 50% growth inhibition com-
pared with untreated control cells.
b A2780 (Human ovarian carcinoma), G401 (human kidney carci-
noma), HT29 (human colorectal carcinoma), H460 (human lung
carcinoma), L1210 (murine leukemia).
9. Ghosh, S.; Schroeter, D.; Paweletz, N. Exp. Cell Res.
1996, 227, 165.
10. Roberge, M.; Tudan, C.; Hung, S. M.; Harder, K. W.;
Jirik, F. R. Cancer Res. 1994, 54, 6115.
11. Cheng, A.; Balczon, R.; Zuo, Z.; Koons, J. S.; Walsh,
A. H.; Honkanen, R. E. Cancer Res. 1998, 58, 3611.
12. McCluskey, A.; Bowyer, M. C.; Collins, E.; Sim, A. T. R.;
Sakoff, J. A.; Baldwin, M. L. Bioorg. Med. Chem. Lett.
2000, 10, 1687.
In view of the anticancer activity and clinical use of
cantharidin, norcantharidin and fostriecin we also con-
ducted cytotoxicity studies in a number of tumour cell
lines, and the results from these studies are shown in
Table 2.
Growth inhibition was evaluated using the MTT
assay.15 From Table 2 it was apparent that cantharidin
(1) and norcantharidin (2) were potent growth inhibi-
tory compounds in all cell lines tested.2 Of the synthe-
sized analogues, compounds 4–15 displayed minimal
growth inhibition with GI50s ranging from 80 mM to
>100 mM. However, compounds 3 and 16 showed
strong growth inhibition with GI50 values comparable
to that of norcantharidin. Interestingly, compound 16
was the most potent PP2A inhibitor, suggesting that this
was the mode of action leading to impaired cellular
replication. Compound 3 on the other hand was less
potent at inhibiting PP1/PP2A activity in vitro, how-
ever, the ester linkage in this compound is susceptible to
intracellular enzymatic cleavage resulting in the poten-
tial production of a more potent intracellular PP1/PP2A
inhibitor. Nonetheless, alternative modes of cell growth
inhibition for this analogue can not be excluded. Simi-
larly, variations in cellular uptake, membrane perme-
ability, drug stability and drug metabolism may account
for the lack of cell growth inhibition in those com-
pounds that were moderate PP2A inhibitors such as
compounds 9, 10, 13, and 14.
13. Wang, G.-S. J. Ethnopharmacol. 1989, 26, 147.
14. Liu, X.-H.; Balzsek, I.; Comisso, M.; Legras, S.; Marion,
S.; Quittet, P.; Anjo, A.; Wang, G.-S. Eur. J. Cancer 1995,
31A, 953.
15. (a) McCluskey, A.; Taylor, C.; Quinn, R. J.; Suganuma,
M.; Fujiki, H. Bioorg. Med. Chem. Lett. 1996, 6, 1025. (b)
Enz, A.; Zenke, G.; Pombo-Villar, E. Bioorg. Med. Chem.
Lett. 1997, 7, 2513. (c) Sodeoka, M.; Baba, Y.; Kobayashi,
S.; Hirukawa, N. Bioorg. Med. Chem. Lett. 1997, 7,
1833. (d) Tatlock, J. H.; Linton, M. A.; Hou, X. J.;
Kissinger, C. R.; Pelletier, L. A.; Showalter, R. E.;
Tempczyk, A.; Villafranca, J. E. Bioorg. Med. Chem. Lett.
1997, 7, 1007. (e) McCluskey, A.; Bowyer, M. C.;
Walkom, C.; Ackland, S. P.; Gardiner, E.; Sakoff, J. A.
Bioorg. Med. Chem. Lett. 2001, 11, 2941. (f) McCluskey,
A.; Keane, M. A.; Walkom, C.; Bowyer, M. C.; Sim,
A. T. R.; Young, D. J.; Sakoff, J. A. Bioorg. Med. Chem.
Lett. 2002, 12, 391.
16. Aggen, J. B.; Humphrey, J. M.; Gauss, C.-M.; Huang,
H.-B.; Nairn, A. A. C.; Chamberlin, A. R. Bioorg. Med.
Chem. 1999, 7, 543.
17. Tatlock, J. H.; Linton, M. A.; Hou, X. J.; Kissinger,
C. R.; Pelletier, L. A.; Showalter, R. E.; Tempczyk, A.;
Villafranca, J. E. Bioorg. Med. Chem. Lett. 1997, 7, 1007.
18. The Diels–Alder cycloadditions giving the respective 5,6-
dehydronorcantharidin ring systems, and in some cases
subsequent catalytic hydrogenations, were carried out
essentially as described in ref 16. Half-acids were prepared
by treating the appropriate anhydrides directly with alco-
hols or amines. Starting materials, reagents, reaction
times, and yields for each analogue are summarised
below: 3: Prepared by the literature procedure, ref 17. 4:
This study clearly illustrates the ability to produce
modified cantharidin and norcantharidin analogues
while maintaining protein phosphatase inhibitory activ-
ity and cell growth inhibition. This study provides the
basis for further development of this class of protein
phosphatase inhibitors for the treatment of malignancy.

M. E. Hart et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1969–1973
1973
From 2; benzyl alcohol, THF, DMAP, 60 ꢀC, 31% yield.
5: From 14; TMSCHN2, EtOAc/MeOH, 0 ꢀC, 66%
yield. 6: Prepared by the general procedure described in
the text, from 3-cyanomethylfuran and maleic anhydride;
19% yield. 7: From 8; H2, Pd/C, THF, 20 ꢀC, 100% yield.
8: Prepared by the general procedure described in the text,
from N-acetyl-2-(aminomethyl)-furan and maleic anhy-
dride; 23% yield. 9: From 2; methyl m-aminobenzoate,
THF, 20 ꢀC, 88% yield. 10: From 11; LiOH in aqueous
THF, À5 ꢀC, 35% yield. 11: From 5,6-dehydro-
norcantharidin; methyl m-aminobenzoate, THF, 20 ꢀC, 24
h, 88% yield. 12: From 5,6-dehydronorcantharidin; ethyl
p-aminobenzoate, THF, 20 ꢀC, 48 h, 83% yield. 13: From
2; ethyl p-aminobenzoate, THF, 20 ꢀC, 48 h, 93% yield.
14: From 2; p-chloroaniline, CH2Cl2, Et3N, 20 ꢀC, 12 h,
80% yield. 15: From 5,6-dehydronorcantharidin; 2 equiv
methylamine, THF, 20 ꢀC, 12 h, 100% yield. 16: From
5,6-dehydronorcantharidin; 2 equivdimethylamine, THF,
20 ꢀC, 12 h, 100% yield.
DMSO and subsequent dilutions were made in distilled
deionised H20. Enzyme dilutions were made with buffer
containing 20 mM MOPS, pH 7.5, 0.15M NaCl, 60 mM
2-mercaptoethanol,
1 mM MgCl2, 2 mM EGTA,
0.1 mM MnCl2, 1 mM DTT, 10% glycerol and 0.1 mg/
mL serum albumin. A dose–response curve of percentage
enzyme activity versus drug concentration was produced
from which an IC50 value was calculated indicating the
concentration of drug required to inhibit enzyme activity
by 50%. Data represent the mean (ÆSEM) IC50 of three
independent replicates. Cell culture and stock solutions.
Stock solutions were prepared as follows and stored at
À20 ꢀC: Cantharidin (Biomol, USA) and cantharidin
analogues as 5 mM solutions in DMSO. All cell lines were
cultured at 37 ꢀC, under 5% CO2 in air. The A2780
(human ovarian carcinoma) and HT29 (human colon
carcinoma) cell lines were maintained in DMEM (Trace
Biosciences, Australia) supplemented with 10 mM sodium
bicarbonate. L1210 (murine leukaemia) cells were main-
tained in RPMI 1640 (Trace Biosciences), while G401
(human kidney carcinoma), were maintained in McCoys
(Trace Biociences). The H460 (human lung carcinoma)
cell line was maintained in RPMI 1640 supplemented with
glucose and pyruvate. All culture media was further sup-
plemented with foetal bovine serum (10%) penicillin (100
IU/mL), streptomycin (100 mg/mL), and glutamine (4
mM). Cells were passaged every 3–7 days and all cell lines
were routinely tested and found to be mycoplasma free.
Cytotoxicity assay. Cells in logarithmic growth were
transferred to 96-well plates. Cytotoxicity was determined
by plating cells in triplicate in 100 mL medium at a density
of 2,500–3,500 cells/well for all cell lines. On day 0, (24 h
after plating) when the cells were in logarithmic growth,
100 mL medium with or without the test agent was added
to each well. After drug exposure growth inhibitory effects
were evaluated using the MTT (3-[4,5-dimethyltiazol-2-yl]
2,5-diphenyl-tetrazolium bromide) assay and absorbance
read at 540 nm The GI50 was the drug concentration at
which cell growth is 50% inhibited based on the difference
between the optical density values on the day 0 and those
at the end of drug exposure (Sakoff, J. A.; Ackland, S. P.
Cancer Chem. Pharm. 2000, 46, 477.)
19. Protein phosphatase inhibition: A non-radioactive in vitro
assay detailed by Gupta was adopted to measure PP1 and
PP2A enzyme activity in the presence of inhibitor drugs
(Gupta, V.; Ogawa, A. K.; Du, X.; Houk, K. N.; Arm-
strong, R. W. J. Med. Chem. 1997, 40, 3199.). Serine/
threonine protein phosphatase assay kit, purified PP1
(rabbit skeletal muscle), PP2A (human red blood cells)
and a hexapeptide (Lys-Arg-pThr-Ile-Arg) substrate was
purchased from Upstate Biotechnology (Lake Placid,
NY). The concentration of PP2A, PP1 and substrate used
in the assay was 0.3 mU/well, 30 mU/well, and 200 mM,
respectively. The reactions were initiated by addition of
substrate (5 mL) to a mixture containing enzyme (5 mL),
reaction buffer (10 mL; 50 mM Tris–HCL, pH 7.0, 100
mM CaCl2), and inhibitor (10 mL), producing a total
reaction volume of 30 mL/well and incubated at room
temperature for 60 min. Immediately prior to addition of
substrate, the enzyme and inhibitor were preincubated for
10 min. Reactions were halted via addition of a malachite
green solution (50 mL), and absorbance readings were
taken at 650 nm after 10 min development time. Samples
were blanked against wells containing enzyme (5 mL), and
buffer (25 mL). Initial inhibitor dilutions were made in