Haloenol pyranones and morpholinones as antineoplastic agents of prostate cancer

Article abstract of DOI:10.1016/j.bmcl.2012.05.038

Haloenol pyran-2-ones and morpholin-2-ones were synthesized and evaluated as inhibitors of cell growth in two different prostate human cancer cell lines (PC-3 and LNCaP). Analogs derived from l- and d-phenylglycine were found to be the most effective antagonists of LNCaP and PC-3 cell growth. Additional studies reveal that the inhibitors induced G2/M arrest and the (S)-enantiomer of the phenylglycine-based derivatives was a more potent inhibitor of cytosolic iPLA₂β.

Full text of DOI:10.1016/j.bmcl.2012.05.038

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4854–4858
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Haloenol pyranones and morpholinones as antineoplastic agents
of prostate cancer
⇑
Jason N. Mock, John P. Taliaferro, Xiao Lu, Sravan Kumar Patel, Brian S. Cummings, Timothy E. Long
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602-2352, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Haloenol pyran-2-ones and morpholin-2-ones were synthesized and evaluated as inhibitors of cell
growth in two different prostate human cancer cell lines (PC-3 and LNCaP). Analogs derived from
and -phenylglycine were found to be the most effective antagonists of LNCaP and PC-3 cell growth.
Additional studies reveal that the inhibitors induced G2/M arrest and the (S)-enantiomer of the phenyl-
glycine-based derivatives was a more potent inhibitor of cytosolic iPLA2b.
Published by Elsevier Ltd.
Received 5 April 2012
Revised 4 May 2012
Accepted 8 May 2012
Available online 17 May 2012
L
-
D
Keywords:
Haloenol
Pyranones
Morpholinones
Phospholipase A2
Prostate cancer
Haloenol pyranones1 are mechanism-based inhibitors of serine
proteases due to their ability to alkylate enzyme active sites fol-
The effects of iPLA2c and iPLA2b on cell signaling and prolifera-
tion have recently been studied by enantiomer-based inhibition6,14
strategies using (R)- and (S)-BEL, respectively (Fig. 2). The mecha-
nisms involved in their selectivity are currently under study
although it was demonstrated that LNCaP and PC-3 prostate cancer
cells display moderate increases in chemosensitivities to racemic
BEL compared to the individual enantiomers.6 These results
suggest that the (R)- and (S)-conformers could be acting in a
lowing ring hydrolysis and unmasking of a reactive
a-haloketone
functionality (Fig. 1). To date, the most evaluated of these inhibi-
tors is bromoenol lactone, (E)-6-(bromomethylene)tetrahydro-3-
(1-naphthalenyl)-2H-pyran-2-one (4) or BEL. Interestingly, the
popularity of BEL stems not as a deactivator of serine proteases,
but rather for its ability to inhibit Ca2+-independent phospholipa-
ses A2 (iPLA2), which are responsible for the catabolism of mem-
brane glycerophospholipids. Over the last 20 years, BEL has
enabled researchers to probe the role of iPLA2 in pathologies
involving oxidative stress and inflammation including cardiovas-
cular,2 Alzheimer’s3 and Parkinson’s diseases,4 diabetes mellitus,5
and more recently, carcinogenesis.6,7
Cys
Cys
His
Ser
His
Ser
Asp
O
O
Nu
X
Asp
Nu
O
R
O
H
HO
O
X
Mammalian cells possess multiple isoforms of iPLA2.8 The most
studied are cytosolic iPLA2b, (Group VIA-1 and A-2 PLA2) and the
R
membrane localized iPLA2c (Group VIB PLA2), which together gov-
ern the release of fatty acids and 2-lysophospholipids from mem-
brane phospholipids. For many years, phospholipid remodeling8,9
was thought to be the only function of these enzymes; however,
beginning in the 1990s researchers began finding evidence that
iPLA2 participates in cell signaling,10 proliferation,11 and death.4,12
It was established that the products arising from the breakdown of
phospholipids functioned as signaling molecules for promoting cell
growth and that the enzymes responsible for generating the lipids
(i.e., PLA2) are in greater abundance in carcinoma cells.13
X = Cl, Br, I
tautomerization
Cys
His
Cys
His
Ser
Ser
O
Nu
OH
Asp
Asp
Nu
H2O
-HX
O
O
HO2C
R
O
R
X
Nu = SH, NH (irreversible); CO2H (reversible)
Figure 1. Mechanism of serinase inhibition by haloenol pyran-2-ones.
⇑
Corresponding author. Tel.: +1 706 542 8597; fax: +1 706 542 5358.
E-mail address: tlong@rx.uga.edu (T.E. Long).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.05.038

J. N. Mock et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4854–4858
4855
O
O
O
O
Br
O
R
O
Br
X
*
N
R
S-BEL
(iPLA2β selective)
haloenol
morpholin-2-one
R-BEL
(iPLA2γ selective)
Figure 2. Chemical structures of BEL and haloenol morpholinones.
X
N
O
O
HO
O
O
O
O
X
O
O
X
aq. K2CO3
DCM, r.t., 3 h
X = Br, I
1
2a
(E)- X = Br
(E)-
2b
I
X =
HO
Ph
O
HO
Ph
O
O
Br
NBS
Br
aq. K2CO3
LDA, THF
-10 oC - r.t., 17 h
Ph
DCM, r.t., 6 h
-3
rac
4
rac-(E)-
Scheme 1. Synthesis of unsubstituted and monophenyl BEL analogs.
synergistic manner as cell growth inhibitors. The studies further
established that enantiomers of haloenol pyranones may be used
to selectively and pharmacologically inhibit iPLA2c, iPLA2b, and
possibly other enzymes involved in critical cell processes. In this
Letter, we report on the antineoplastic activities of haloenol pyr-
an-2-one analogs of BEL against prostate cancer. In addition, the
evaluation of novel haloenol morpholin-2-ones constructed from
Br
R
R
HClO4
*
*
H2N
CO2tBu
tBuOAc
r.t., 20 h
LiOH.H2O
MgSO4, MeCN
H2N
CO2H
r.t., 15-18 h
- Phg = (S)-5 R = Ph
-Phg = (R)-5 R = Ph
L-Pha = (S)- R = Bn
Gly = 7 R = H
L
(S)-8 R = Ph
8
D
(R)- R = Ph
6
(S)-9 R = Bn
10
L- and D-amino acids and their inhibitory effects on the cell cycle
R = H
and iPLA2 activity are described.
To evaluate whether analogs of BEL could have similar inhibi-
tory effects on iPLA2 and prostate cancer growth, we set forth to
O
O
N
X
R
1. TFA, DCM
2. NBS or NIS
synthesize various haloenol pyran-2-ones from
a-substituted and
*
t
N
CO2 Bu
R
unsubstituted acetylenic acids. Standard E-specific haloenol lact-
onization procedures1,15 with N-halosuccinimides (X = Br, I) were
used to generate the pyranone analogs (Scheme 1). In the case of
the phenyl analog 4, the acid precursor 3 required preparation
from phenylacetic acid and 4-bromobut-1-yne using classical eno-
late chemistry.1g Subsequent attempts to separate the (R)- and (S)-
enantiomers of lactone 4 by chiral HPLC were unsuccessful, which
led us to consider the use of chiral pool amino acids to construct
novel iPLA2 inhibitors containing a E-haloenol morpholin-2-one
framework (Fig. 2).
aq. K2CO3, DCM
r.t., 4-8 h
(S,E)-14a R = Ph, X = Br
(R,E)-14b R = Ph, X = Br
(S)-11 R = Ph
(R)-11 R = Ph
15
(S,E)- R = Ph, X = I
12
(S,E)-16 R = Bn, X = Br
(E) -17 R = H, X = Br
(S)-
R = Bn
13 R = H
Scheme 2. Synthesis of N-propargyl haloenol morpholin-2-ones 14–17.
L- and D-phenylglycine (Phg), L-phenylalanine (Pha), and glycine
(Gly) were chosen as base materials to perform the asymmetrical
synthesis of morpholinone analogs (Scheme 2). Protected tert-
butyl esters forms of the amino acids were first prepared from
tert-butyl acetate16 then converted to the the corresponding N,N-
Minimum inhibitory concentrations (IC50s) were determined by
MTT staining for the haloenol pyranones (2, 4) and morpholinones
(14–17, 20) against LNCaP cells, and the more resistant PC-3 hu-
man prostate cancer cell line. With racemic BEL as a haloenol stan-
dard, IC50 measurements were taken at 24, 48, and 72 h (Table 1).
BEL was found to inhibit growth in a time-dependent manner at 5–
propargyl
a
-amino esters 11–13.17 Following deprotection of the
carboxylic acid, bromo- and iodoenol morpholin-2-one analogs
14–17 were generated in 6–23% yield under the conditions
described for pyranones 2 and 4.
13 and 14–34 lM in LNCaP and PC-3 cells, respectively, over 72 h,
which corroborated previous findings.6 Activity comparison of BEL
The synthesis of additional
L
-Phg-based analogs was also
to pyranones 2 revealed that the unsubstituted analogs were
attempted from the monopropargyl intermediate 18 (Scheme 3).
Benzylation of the secondary amine followed by acid deprotection
and cyclization gave the corresponding N-benzyl bromoenol mor-
pholin-2-one 20. Efforts to prepare the unsubstituted analog 21
were unsuccessful however, which was attributed to chemical
instability of the N-protio ring system.
equally efficacious inhibitors at 5–10 and 14–32
sponding cell lines. For the -substituted phenyl analog 4, slightly
enhanced activities were observed with IC50s ranging from 6 to
27 M against PC-3 cells.
The morpholinones analogs similarly demonstrated antineo-
plastic activity with IC50s reaching for the Phg-based
lM for the corre-
a
l
3 lM

4856
J. N. Mock et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4854–4858
mented activity of (R)-14b was attributed in part to higher prote-
olytic susceptibility (e.g., chymotrypsin) that the L-Phg-based (S)-
14a may have in the cell.
O
O
1. TFA, DCM
2. NBS
Br
N
H
t
Other haloenol morpholinones were found to have weaker
inhibitory activities including the -Pha- and Gly-derived analogs
aq. K2CO3, DCM
r.t., 9 h
N
H
CO2 Bu
L
16 and 17, respectively. Surprisingly, chemosensitivity for the iod-
oenol derivative 15 was also considerably lower than its bromo-
(S,E)-21
(S)-18
not formed
BnBr
enol counterparts 14. Conversely, the N-benzyl
analog 20 proved to be the most potent antagonist in the study
(IC50 1–4 M). The compound demonstrated rapid and sustained
L-Phg-based
LiOH.H2O
MgSO4, MeCN
r.t., 18 h
l
inhibitory effects on cell proliferation for both LNCaP and PC-3 cells
over the 72 h evaluation period.
To determine if growth inhibition was due to cytostatic or cyto-
toxic effects by the antagonists, cell viability was assessed by
phase-contrast microscopy.18 Comparisons of morphology were
made by visual inspection of LNCaP cells following 72 h treatment
O
O
N
1. TFA, DCM
2. NBS
Br
N
CO2tBu
aq. K2CO3, DCM
r.t., 5 h
with rac-BEL, rac-4, (S)-14a, and (R)-14b (Fig. 3). Exposure to 5 lM
of BEL and its monophenyl analog 4 induced little to no morpho-
logical changes in cell shape, differentiation, and death compared
to the vehicle (DMSO) control. For the morpholinone analogs 14,
apoptosis and/or necrosis was evident at the same concentrations
particularly for the (R)-enantiomer. It was concluded from these
microscopic images that prostate cancer cells had greater chemo-
sensitivity to haloenol morpholinones than to the analogous haloe-
nol pyranones which corroborated the IC50 data.
The inhibitory effects by rac-BEL, pyranone 4, and morpholinon-
es 14 were additionally assessed my monitoring changes in the cell
cycle by flow cytometry with propidium iodide6b (Fig. 3). Moderate
increases of LNCaP cell counts in the G1 phase were observed fol-
(S)-19
(S,E)-20
Scheme 3. Synthesis of N-benzyl bromoenol morpholin-2-one 20.
Table 1
IC50s (
lM) against human prostate cancers after 24, 48, and 72 h exposure to haloenol
inhibitorsa
Compound
LNCaP
48
PC-3
48
24
72
24
72
rac-BEL
2a
2b
rac-4
(S)-14a
(R)-14b
(S)-15
(S)-16
17
13
10
9
31
8
5
5
5
5
3
6
23
26
29
4
9
5
7
4
3
3
20
32
28
3
34
19
32
27
15
8
21
33
13
4
26
23
15
10
13
6
21
57
10
1
14
14
16
6
5
3
25
39
7
lowing 24 h treatment with 5 and 10 lM of the test compounds. It
is believed that the elevated G1 levels led to the decrease in S and
G2/M phase cell percentages and the effects were greatest for 14a
and 14b, which induced complete cell cycle arrest at 10
lM. Like-
6
wise, on comparison to cultures treated with the 5 M of the inhib-
l
26
41
25
3
itors, the increase of cells residing in S phase may have been due to
the lack of cells entering the G2/M phase. These results further sug-
gest that the cytotoxic effects of morpholinone-based analogs may
be the result of DNA hypoploidy, which is associated with DNA
fragmentation and apoptosis. Examples of agents that block mito-
sis by inhibiting chromosome replication include DNA alkylating
agents (e.g., nitrogen mustards) and antagonists of glutathione S-
(S)-20
4
a
Data represent the calculated IC50 using data assessed from 3–5 experiments
ran in duplicate using separate passages of cells assessing alteration in MTT
staining.
transferase (e.g.,
a
-chloroacetamides19), which protect cells from
oxidative DNA damage.
Lastly, rac-BEL, pyranone 4, and morpholinones 14 were evalu-
ated for their ability to inhibit iPLA2b from rat kidney. Cytosolic
derivatives 14 (Table 1). The inhibitors also appeared to be more
rapid-acting antagonists of prostate cancer growth compared to
BEL and its phenyl pyranone analog 4. Moreover, activity compar-
ison of the enantiomers revealed that (R)-14b was a more effective
fractions were treated for 0.5 h with 0–100 lM of the compounds
prior to inoculation with the arachidonoyl thio-phosphatidylcho-
inhibitor than (S)-14a particularly against PC-3 cells (IC50 3–8
lM).
line, a hydrolysable thioester-containing probe of PLA2 activity.6b
As a compound derived from the unnatural -form of Phg, the aug-
D
Figure 3. Changes in morphology (left-40Â magnification) and cell cycle (right) of LNCaP cells following treatment with rac-BEL, rac-4, (S)-14a, and (R)-14b.

J. N. Mock et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4854–4858
4857
Both rac-BEL and its phenyl-substituted analog 4 demonstrated
nearly identical efficacy to inhibit the enzyme in a concentration-
dependent manner (Fig. 4). Inhibitory activity was also noted for
(S)-14a but to a lesser degree compared to pyranone-based antag-
onists. Little to no effects on iPLA2b activity was observed for (R)-
14b, which correlates to earlier findings6,14 that the (S)-enantiomer
of BEL selectively inhibits cytosolic iPLA2b while (R)-BEL possesses
higher affinity for microsomal iPLA2c.
In summary, haloenol pyran-2-ones were found to be effica-
cious inhibitors of prostate carcinoma cell growth and iPLA2b activ-
ity however, as with BEL, a definitive correlation could not be
made. Novel haloenol morpholin-2-ones constructed asymmetri-
cally from chiral amino acids were also discovered to be antago-
nists of cell proliferation. Differences in the effects on the cell
cycle and iPLA2b activity suggested that the morpholinone analogs
14 may have a greater capacity to directly or indirectly cause DNA
damage. Glutathione S-transferase which has a role in protecting
DNA from oxidative damage is known to be inhibited by haloenol
lactones20 and could be a primary or secondary target for the Phg-
based derivatives. Finally, during the course of these studies it be-
came apparent that the chemical instability of the haloenol pyra-
nones and morpholinones would likely preclude them from being
viable drug candidates for prostate cancer. Their use as research
tools in the study of tumorigenesis and validation of new therapeu-
tic targets may be of great value though to the drug discovery
community.
Acknowledgments
Financial support was generously provided by the College of
Pharmacy at the University of Georgia and in part by a Georgia
Cancer Coalition Distinguished Scholar Grant and a NIH NIBIB
(EB08153) to B.S.C. In addition, author J.N.M. gratefully acknowl-
edges support from the American Foundation of Pharmaceutical
Education (AFPE) and Achievement Rewards for College Scientists
(ARCS).
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