Bioactive 4-substituted-6-methyl-2-pyrones with promising cytotoxicity against A2780 and K562 cell lines.

Article abstract of DOI:10.1016/S0960-894X(02)00824-7

Bioactive synthetic 4-substituted-6-methyl-2-pyrones are reported. Various 4-substitutents have been incorporated using Pd-catalysed carbon-carbon bond coupling procedures. Preliminary screening of the 2-pyrones against human ovarian carcinoma (A2780) and

Full text of DOI:10.1016/S0960-894X(02)00824-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3509–3513
Bioactive 4-Substituted-6-methyl-2-pyrones with Promising
Cytotoxicity against A2780 and K562 Cell Lines
Lester R. Marrison,a Julia M. Dickinsona and Ian J. S. Fairlamba,b,
*
aDepartment of Chemistry and Materials, John Dalton Building, The Manchester Metropolitan University, Chester Street,
Manchester M1 5GD, UK
bDepartment of Chemistry, University of York, Heslington, York YO10 5DD, UK
Received 4 September 2002;revised 27 September 2002;accepted 3 October 2002
Abstract—Bioactive synthetic 4-substituted-6-methyl-2-pyrones are reported. Various 4-substitutents have been incorporated using
Pd-catalysed carbon–carbon bond coupling procedures. Preliminary screening of the 2-pyrones against human ovarian carcinoma
(A2780) and human chronic myelogenous leukaemia (K562) cell lines show that 4-alkynyl-6-methyl-2-pyrones have excellent
potential as anticancer agents. The pyrones demonstrate broad spectrum antimicrobial activities.
# 2002 Elsevier Science Ltd. All rights reserved.
The 2-pyrone sub-unit1 is an important synthetic build-
ing block2 found in numerous biologically active natural
products,3 for which there has been considerable recent
interest.4 Of interest to us are several reports on cyto-
toxic pyrones from natural sources.5 The Bufadienolides
(1) provide an excellent example with a diverse range of
biological activities (Fig. 1).6
and alkynyl substituents was required. For this we
focused our attention on Pd-catalysed C–C bond form-
ing processes, as we felt that the range of substituents
that could be incorporated would be greater and
accomplishable in higher yields than the more laborious
traditional methods for introducing substituents into
the pyrone ring system. There are surprisingly few
reports on direct Pd-catalysed coupling of nucleophiles,
such as boranes, boronic acids, benzodioxaboroles and
alkynylcopper reagents (generated under Songashira
conditions), onto the electrophilic pyrone ring system.
There are two reports on Pd-catalysed coupling of alkyl
One particular drawback of such natural products is
their molecular complexity and low yields from natural
extraction/isolation procedures. We therefore had a
desire to synthesise much simpler 2-pyrone systems with
a view to identifying promising: (1) bioactivity against
cancer cells;and (2) antimicrobial activity. A potential
and promising field of study is in the application of
antimicrobial pyrones for use in agriculture, as pesti-
cides or herbicides. Fusapyrone 27 is a recent example of
a 2-pyrone with considerable antifungal activity, low
phytotoxicity and mycotoxicity.
We herein report on the synthesis and biological eval-
uation of relatively simple 4-alkyl/aryl/alkenyl/alkynyl-
6-methyl-2-pyrones 3.
An efficient synthetic methodology that allows for the
facile introduction of a plethora of alkyl, aryl, alkenyl
*Corresponding author. Tel.: +44-1904-434091;fax: +44-1904-
432516;e-mail: ijsf1@york.ac.uk
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00824-7

3510
L. R. Marrison et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3509–3513
and alkynyl zinc reagents to 4-bromo-6-methyl-2-pyr-
one.8 In particular, an efficient synthesis to the cockroach
sex pheromone, Supellapyrone (5-(20R,40R-dimethylhep-
tanyl)-3-methyl-2-pyrone) has been reported using an
alkylzinc reagent as the key step, which allowed the syn
stereochemistry of the alkyl chain to be deduced.8a,b
mandatory base in a THF–H2O solvent mixture, which
allowed the reactions to be run at 25 ꢀC (Table 1). The
yields of cross-coupled products (5a–h) were satisfactory
and thus at this point we did not investigate more effi-
cient catalysts or reaction conditions.
Coupling with arylboronic acids
We chose 4-bromo-6-methyl-2-pyrone 4 (available in
79% yield using PBr3 in DMF at 70 ꢀC from com-
mercially available 4-hydroxy-6-methyl-2-pyrone) as
the starting material for the coupling reactions
(Scheme 1).
The Suzuki coupling of 4 with several boronic acids
proceeded well using Pd(0) based catalysts, such as tet-
rakis-(triphenylphosphine)palladium(0) [(PPh3)4Pd] and
a palladium(II)acetate [Pd(OAc)2]/triphenylphosphine
combination. The latter catalyst proved more versatile
and less sensitive to air. The bases generally used in
Suzuki couplings are NaOEt and NaOH, but these were
far to strong for the 2-pyrone sub-unit to withstand, due
to basic hydrolysis.
Coupling with trialkylboranes
The synthesis of 4-alkyl-6-methyl-2-pyrones (5) were
initially envisaged as being easily accessible from alkyl-
boronic acids and alkylbenzooxaboroles. However, sev-
eral Pd-catalysts and conditions (solvents, base and
reaction temperature) were unsuccessful. To our delight,
we were able to couple trialkylboranes with 4 using [1,10-
bis(diphenylphosphino)ferrocene]dichloropalladium(II)
[Pd(dppf)Cl2] as the catalyst, thallium carbonate as a
We found that use of Na2CO3 was of paramount
importance to the yields of 6. In Table 2, coupling of
several boronic acids can be found. The electron-
donating arylboronic acids (entries 2 and 4, Table 2)
provided the coupled products in generally good yields.
Changing the ligand to trifurylphosphine improved the
yields. Although little problems were seen with the
chloro-substituent (entry 3, Table 2), changing to the
more electron withdrawing nitro and formyl sub-
stituents had a dramatic effect on the yields (entries 5
and 6, Table 2). For the latter, the P(furyl)3 ligand
improved the yield to 43% from 20%.
Coupling with alkenylboronic acids
The initial investigations into coupling alkenylbenzo-
dioxaboroles with 4 under Pd(0) catalysis gave the cou-
pled products in 30–40% yields. However, we felt these
reactions were cumbersome and the air-sensitive benzo-
dioxaboroles made them unattractive. On the other
hand, coupling of 4 to alkenylboronic acids were much
more facile and yields of the 4-alkenyl-2-pyrones
improved dramatically (Table 3). As for the arylboronic
acids, use of Na2CO3 proved a mandatory base. It is of
interest that we have so far been unable to synthesise
.
Scheme 1. Pd-catalysed C–C bond couplings of 4: (i) BH3 THF,
alkene, 0 ꢀC then Pd(dppf)Cl2, Tl2CO3, THF/H2O, 25 ꢀC;(ii) Pd(OAc) 2,
PPh3, arylboronic acid, Na2CO3, benzene, Á;(iii) Pd(OAc) 2, PPh3
RCH¼CHM (E), Na2CO3, benzene, Á;(iv) Pd/C, PPh 3, CuI, Et3N,
CH3CN, Á.
Table 1. Coupling of various alkenes with 4a
Table 2. Coupling of various arylboronic acids with 4a,b
Entry
Pyrone (R)
Yield
Entry
Pyrone (R)
Yield
1
2
3
4
5
6
H–, 6a
p-CH3–, 6b
p-Cl–, 6c
p-CH3O–, 6d
m-NO2–, 6e
p-CHO–, 6f
56 (81)
52 (76)
53
62 (86)
0
20 (43)
1
2
3
4
5
6
7
8
H, 5a
56
60
50
46
54
41
59
32
CH3CH2–, 5b
CH3(CH2)2–, 5c
CH3(CH2)3–, 5d
CH3(CH2)4–, 5e
CH3(CH2)5–, 5f
Ph–, 5g
aConditions: (i) Pd(OAc)2 (6 mol%), PPh3 (18 mol%), arylboronic
acid, Na2CO3, benzene, Á, 6 h.
bUsing P(furyl)3 (18 mol%) as the ligand in place of PPh3, yields in
parentheses.
CH3CH2O–, 5h
a
ꢀ
.
Conditions: (i) BH3 THF, alkene, 0 C, 1 h;(ii) Pd(dppf)Cl
mol%), Tl2CO3, THF/H2O, 25 ꢀC.
(5
2

L. R. Marrison et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3509–3513
3511
these analogues using Heck coupling of 4 with alkenes
under a variety of standard conditions.
using an in vitro cell culture system (MTT assay).10 The
assay itself is based on the reduction of 3-(40,50-dimethyl-
thiazol-20-yl)-2,5-diphenyl-tetrazolium bromide (MTT,
yellow colour) by mitochondrial dehydrogenases of
metabolically active cells to a purple-blue formazan.
The results are shown in Table 5. Comparable potent
inhibitors against these cell lines have been reported.11
Coupling with alkynes (Sonogashira)
We have recently found that terminal acetylenes couple
to 4 using Sonogashira coupling. Exploration of a range
of catalyst systems led to the surprising finding that Pd/C
efficiently catalyses the coupling of 4 with terminal acet-
ylenes. In Table 4 can be found the yields of the 4-alky-
nyl-6-methyl-2-pyrones from several terminal acetylenes.
From the data in Table 5, it is evident that the anti-
proliferative activity is variously manifested in the two
cell lines and it is possible to discern some quite promi-
nent structure–activity relationships. The compounds
that were not active at IC50 <50 mM were those which
contained the 4-alkyl and 4-alkenyl substitutents (5a–d,
5g–h, 7c–e), although some activity was observed for the
heptyl derivative 5e which had an IC50 (K562) of 28.7
mM and IC50 (A2780) of 41.8 mM. The most potent
compounds in this series were the 4-alkynyl derivatives
(8b–g and 8k). The most potent derivative against A2780
was phenylethynyl derivative 8b with an IC50 of 1.8 mM.
Compound 8b was also active against K562 (IC50=4.0
mM). Increasing the alkynyl chain from pentynyl through
to a heptynyl chain (8c–e) sees an improvement in activ-
ity against the K562 cell line, peaking at 8e.
The TMS derivative 8a was synthesised with a view to
accessing the free alkyne 8k. Silyl cleavage is best per-
formed using tetrabutylammonium fluoride (TBAF) in
THF (1 M) at ꢁ78 ꢀC. After 2 h 8k is isolated in 80%
yield.9
Biological Results
The growth inhibitory activities of the 2-pyrones were
determined in the A2780 human ovarian carcinoma and
K562 human chronic myelogenous leukaemia cell lines
By extending the alkynyl chain by one carbon to 8f, we
see a fall off in activity. A slightly different trend for the
A2780 cell line is observed, although the activity again
peaks with 8e. A somewhat striking result is observed
by comparing the activities of 8f for both cell lines
[IC50=(A2780) 3.4 mM and (K562) 20.3 mM]. Such dif-
ferences are observed with alkynyl derivatives 8c–f for
both cell lines to a more or lesser degree. Two further
potent analogues, in addition to 8b, are 8g and 8k with
an IC50 (A2780) of 2.1 and 3.1 mM and IC50 (K562) of
4.0 and 2.0 mM, respectively.
Table 3. Suzuki coupling of alkenylboronic acids with 4a
Entry
Pyrone (R)
Yield
1
2
3
4
5
CH3(CH2)2–, 7a
CH3(CH2)3–, 7b
CH3(CH2)4–, 7c
CH3(CH2)5–, 7d
Ph–, 7e
58
66
60
56
27
Table 5. In vitro IC50 cytotoxicity results against A2780 and K562
cell lines10
aConditions: (i) Pd(OAc)2 (6 mol%), PPh3 (18 mol%), alkenylboronic
acid, Na2CO3, benzene, Á, 6 h.
R
A2780 (mM)
K562 (mM)
Ethyl, 5a
Butyl, 5b
Pentyl, 5c
Hexyl, 5d
Heptyl, 5e
2-Phenylethyl, 5g
Ethoxyethyl, 5h
>50
>50
>50
>50
41.8
>50
>50
>50
>50
>50
>50
28.7
>50
>50
Table 4. Sonogashira coupling of terminal alkynes with 4a
Ph, 6a
p-CH3–, 6b
p-Cl–, 6c
p-CH3O–, 6d
p-CHO–, 6f
>50
>50
>50
>50
19.2
>50
>50
>50
>50
26.0
Entry
Pyrone (R)
Yield
1
2
(CH3)3Si–, 8a
Ph–, 8b
82
74
3
4
5
6
CH3(CH2)2–, 8c
CH3(CH2)3–, 8d
CH3(CH2)4–, 8e
CH3(CH2)5–, 8f
THPOCH2–, 8g
p-NH2-Ph–, 8h
p-NHAc-Ph–, 8i
p-NO2-Ph–, 8j
H–, 8k
72
77
79
73
81
21
95
95
Hexenyl, 7c
Heptenyl, 7d
Phenylethenyl, 7e
>50
>50
>50
>50
>50
>50
8
9
10
11
See text
Phenylethynyl, 8b
Pentynyl, 8c
Hexynyl, 8d
Heptynyl, 8e
Octynyl, 8f
1.8
4.8
7.0
2.9
3.4
2.1
3.1
4.0
17.8
15.5
11.1
20.3
4.0
See text
THPOCH2, 8g
Ethynyl, 8k
aConditions: (i), 10% Pd/C (20 wt%), PPh3 (25 wt%), dry Et3N, dry
CH3CN (2.5:1.5), CuI (4 mol%) under N2, 3 h.
2.0

3512
L. R. Marrison et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3509–3513
Table 6. Antimicrobial activities of 4-substituted-6-methyl-2-pyronesa
Compd
Bacillus subtilisb
Escherichia colib
Staphylococcus
aureusb
Candida
albicansc
Saccharomyces
cerevisiaec
Schizosaccharomyces
pombec
5a
5b
5c
5d
5e
5f
0
21 (23.8)
22 (25.0)
22 (25.0)
25 (28.4)
17 (19.3)
22 (25.0)
20 (22.7)
NT
0
0
0
0
0
0
0
0
0
0
0
0
20 (22.7)
23 (26.1)
21 (23.9)
16 (18.2)
19 (21.6)
18 (20.5)
NT
22 (25.0)
26 (29.5)
30 (34.1)
30 (34.1)
10 (11.4)
0
11 (12.5)
16 (18.2)
20 (22.7)
0
0
5 (5.7)
0
14 (15.9)
18 (20.5)
0
5g
5h
0
NT
NT
NT
6a
6b
6c
6d
6f
18 (20.5)
19 (21.6)
18 (20.5)
17 (19.3)
18 (20.5)
20 (22.7)
18 (20.4)
20 (22.7)
20 (22.7)
15 (17.0)
18 (20.5)
0
20 (22.7)
0
18 (20.5)
0
0
0
0
0
0
0
0
0
0
0
0
0
20 (22.7)
14 (15.9)
7a
7b
7c
7d
7e
16 (18.2)
18 (20.5)
18 (20.5)
18 (20.5)
18 (20.5)
19 (21.6)
18 (20.5)
18 (20.5)
20 (22.7)
17 (19.3)
20 (22.7)
0
17 (19.3)
18 (20.5)
0
16 (18.2)
24 (27.3)
21 (23.9)
20 (22.7)
0
0
0
0
0
0
28 (31.8)
40 (45.5)
34 (38.6)
29 (32.9)
18 (20.5)
8b
8c
8d
8e
8f
8g
8i
8j
8k
19 (21.6)
18 (20.5)
17 (19.3)
19 (21.6)
17 (19.3)
20 (22.7)
6 (6.8)
20 (22.7)
22 (25.0)
20 (22.7)
19 (21.6)
17 (19.3)
17 (19.3)
0
0
0
0
0
0
0
0
0
0
37 (42.0)
29 (32.9)
45 (51.1)
50 (56.8)
48 (54.5)
18 (20.5)
0
21 (23.9)
0
11 (12.5)
20 (22.7)
22 (25.0)
0
13 (14.8)
0
21 (23.9)
18 (20.5)
30 (34.1)
16 (18.2)
0
6 (6.8)
17 (19.3)
0
16 (18.2)
0
36 (40.9)
0
40 (45.4)
9 (10.2)
40 (45.5)
30 (34.1)
aEach cellulose disk contained 200 mg of the test compound. NT, not tested. Radii/mm (percent) of inhibition.
bAfter 96 h incubation.
cAfter 48 h incubation.
The aryl derivatives (6a–d) showed no inhibitory activ-
ity at a concentration of <50 mM. However, formyl
substituted derivative 6f demonstrated promising activ-
ity against both cell lines (IC50=(A2780) 19.2 mM and
(K562) 26.0 mM). This result is significant in that it
illustrates the effect of making a simple change in sub-
stituent can have such a dramatic outcome.
The results clearly suggest that the presence of a hydro-
phobic 4-substituent is key to inhibitory activity.
In conclusion we have synthesised a plethora of 4-alkyl/
aryl/alkenyl/alkynyl-6-methyl-2-pyrones using Pd-cata-
lysed coupling procedures. These studies represent the
first comprehensive investigations into such coupling
processes for 4-bromo-2-pyrones and important reac-
tion conditions are given. The 4-alkynyl-6-methyl-2-
pyrones have been identified as promising new cytotoxic
agents against both A2780 and K562 cell lines and
details of significant antimicrobial activities have been
presented. In short the 4-substituted pyrones are ex-
tremely bioactive in a range of species and in due course
we aim to uncover their mode(s) of action.
The antimicrobial results can be seen in Table 6. Potent
control compounds were used for comparison in these
assays.12 We have detailed a comprehensive set of data
for the 2-pyrones against three bacteria and three
yeasts. The assay used was a standard agar diffusion
method as previously described.13
With the exception of compounds 8i and 8j all the pyr-
ones tested are active against B. subtilis and E. coli. The
most potent pyrones were the pentyl and hexyl deriva-
tives 5c and 5d, although both of these derivatives
demonstrate no activity towards S. aureus. The most
potent pyrones against S. aureus by some distance were
8g and 8k. Indeed 8k was also potent towards the
yeasts, C. albicans, S. cerevisiae and S. pombe, whereas
8g showed only modest activity against these yeasts.
With the exception of 8k, insignificant activity was
observed for the pyrones against S. cerevisiae— an
intriguing result! In stark contrast, potent activity was
observed against S. pombe for many of these pyrones.
The most potent analogue was heptynyl derivative 8e.
Acknowledgements
We thank the Patterson Institute for Cancer Research
(Manchester, UK) for cell line testing, in particular Dr
Tim Ward of the PICR Cell Culture Unit for maintain-
ing the cell line and assistance with the bioassay. The
EPSRC (PhD studentship to L.R.M.) and MMU for
funding are gratefully acknowledged. I.J.S.F. thanks the
U.o.Y. for financial support in the form of an IRPF
grant. We gratefully acknowledge a generous loan of
palladium salts from Johnson Matthey PLC.

L. R. Marrison et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3509–3513
3513
References and Notes
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