Arene cis-dihydrodiols: Useful precursors for the preparation of analogues of the anti-tumour agent, 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC)

Article abstract of DOI:10.1016/j.bmcl.2007.07.070

The synthesis of 6-epi-COTC, a diastereoisomer of Streptomyces metabolite 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC), is described. The anti-cancer activities of the novel analogue, in racemic and enantiomerically pure forms, a

Full text of DOI:10.1016/j.bmcl.2007.07.070

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5974–5977
Arene cis-dihydrodiols: Useful precursors for the preparation
of analogues of the anti-tumour agent, 2-crotonyloxymethyl-
(
4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC)
a
a
a
a,
*
Claire L. Arthurs, James Raftery, Helen L. Whitby, Roger C. Whitehead,
b
b
Natasha S. Wind and Ian J. Stratford
a
School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK
b
Received 22 June 2007; revised 18 July 2007; accepted 18 July 2007
Available online 21 August 2007
Abstract—The synthesis of 6-epi-COTC, a diastereoisomer of Streptomyces metabolite 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-tri-
hydroxycyclohex-2-enone (COTC), is described. The anti-cancer activities of the novel analogue, in racemic and enantiomerically
pure forms, are presented.
Ó 2007 Elsevier Ltd. All rights reserved.
2
-Crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclo-
O
O
hex-2-enone (COTC, 1) was isolated in 1975 from
cultures of Streptomyces griseosporeus (Fig. 1). It has
been demonstrated that both 1 and its non-hydroxylated
analogue, 2-crotonyloxymethyl-cyclohex-2-enone (COMC,
O
O
O
O
HO
OH
OH
2
cancer cell lines.
), display potent toxicity towards murine and human
1
–3
COTC
1
COMC
2
4
Extensive investigations by several researchers have
demonstrated that the mechanism of anti-cancer activity
of the cyclohexenones may involve conjugation of gluta-
thione (GSH) to 1 or 2 thereby generating a glutathiony-
lated exocyclic enone of type 3 (Scheme 1): this reaction
can be catalysed by glutathione transferase (GST).
Alkylation of intracellular proteins and/or nucleic acids
by 3 is believed to then lead to cell death. A consequence
of this mechanism of action is the probability that cells
that possess elevated levels of GST/GSH will exhibit
enhanced sensitivity towards exposure to 1 or 2.
Figure 1. Structures of COTC and COMC.
O
GSH / GST
O
O
GS
O
1, 2
3
DNA / protein
RNH
Recently, we initiated a synthetic chemistry programme
designed to prepare analogues of COTC with general
structure 5, wherein the C4, C5 and C6 substituents
represent three loci for structural variation (Fig. 2).
Initial studies of a small array of these analogues have
O
cell death
4
Scheme 1.
indicated that racemic compound 6 is up to four times
more potent than COMC (2) towards lung cancer cell
lines: previously, comparative bioassays by Douglas
Keywords: Glutathione; Glutathione transferase; Anti-tumour; COTC.
*
Corresponding author. Tel.: +44 161 275 7856; fax: +44 161 275
939; e-mail: roger.whitehead@manchester.ac.uk
5
4
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.070

C. L. Arthurs et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5974–5977
5975
O
O
O
O
O
2
O
X
O
Z
O
O
3
1
HO
OH
4
6
5
HO
OH
Y
5
6
7
Figure 2. Analogues of COTC.
and co-workers of 1 and 2 against a range of cancer cell
lines showed 2 to be more potent than 1 in almost all
cases. In order to gain further knowledge concerning
Direct trans-ketalisation of (±)-11 to give (±)-13 proved
to be an inefficient process (Scheme 3). This key trans-
formation could be achieved, however, in acceptable
yield via initial removal of the acetonide group followed
by subsequent protection of the vicinal trans-diequatorial
hydroxyls as their butanediacetal (BDA) derivative.
Variable quantities of another acetal (tentatively
assigned as (±)-14 [15–20%]) together with a minor
amount (ꢀ5%) of methoxylated compound (±)-15 were
also isolated from this two-stage procedure: the latter is
presumed to arise from nucleophilic participation by the
reaction solvent during introduction of the BDA group.
Oxidation of the allylic hydroxyl of (±)-13 proceeded
smoothly to give enone (±)-16 and subsequent introduc-
tion of a hydroxymethyl side-chain was accomplished
using an imidazole-mediated Morita–Baylis–Hillman
3
the importance of relative and absolute stereochemistry,
as well as the extent of hydroxylation, on observed bio-
activity, we have now prepared 6-epi-COTC (7) in race-
6
mic and enantiomerically pure forms. In this Letter, we
provide details of these syntheses as well as the results of
bioassay of the target compounds against lung cancer
cell lines A549 and H460.
The synthesis of (±)-7 commenced with the meso arene-
dihydrodiol 8 derived from enzymatic dihydroxylation
of benzene (Scheme 2). Acetonide formation followed
by face-selective cis-dihydroxylation using Upjohn con-
7
ditions then provided diol (±)-9. We felt, initially, that
1
3,14
selective oxidation of the allylic hydroxyl of (±)-9 to give
enone (±)-10 would provide the basis for an expedient
synthesis of the target compound. Unfortunately, how-
reaction.
for the latter reaction, including the use of alternative
Other conditions were also investigated
1
5
nucleophilic catalysts such as DMAP,
but these
8
ever, oxidation of (±)-9 using either PDC in DMF or
9
DDQ in THF gave the enone in quite disappointing
proved less effective than the imidazole-mediated pro-
cess with regard to the isolated yield of (±)-17 as well
as reproducibility.
yields. We turned, therefore, to an alternative approach
and were pleased to discover that selective protection of
the allylic hydroxyl of (±)-9 as its 2-naphthylmethyl
Crotonylation of the primary hydroxyl of (±)-17 under
standard conditions gave the fully protected target com-
pound (±)-18 (Scheme 4). One of the benefits of Nap
protection for hydroxyl groups is the potential for facile
(
an intermediate stannylene acetal. Minor quantities
Nap) ether could be accomplished in good yield via
1
0
(ꢀ10%) of readily separable regioisomer (±)-12 were
also generated in this reaction.
1
6
deprotection under mildly oxidative conditions. We
were pleased, therefore, to discover that exposure of
(±)-18 to DDQ at rt for 6 h furnished allylic alcohol
(±)-19 in reasonable yield and removal of the BDA
group under standard conditions then gave the target
The structure of (±)-11 was initially assigned by analysis
of the H NMR data of its 4-O-acetyl derivative and
1
confirmation of the relative stereostructure was ulti-
mately gained by X-ray analysis of a crystal of (±)-11
1
7
compound in racemic form. An alternative sequence
of deprotection steps involving acid-catalysed hydrolysis
of (±)-18 to give (±)-20 followed by exposure to DDQ
also proceeded smoothly to give 6-epi-COTC ((±)-7).
1
1,12
obtained from petroleum ether/ethyl acetate (Fig. 3).
OH
The asymmetric synthesis of (ꢁ)-7 required the prepara-
O
O
i), ii)
iii) or iv)
O
18
tion of enantiopure diol (ꢁ)-9. This was accomplished
O
O
from the biotransformation product 21 of iodobenzene.
Formation of the acetonide derivative of 21 followed by
cis-dihydroxylation gave diol (+)-22 and subsequent
OH HO
8
(
±)-9
(±)-10
OH
OH
v)
19
chemoselective reduction of the C–I bond yielded
(
O
O
O
ꢁ)-9 in good overall yield. An identical sequence of
+
reactions to that described above gave the fully pro-
tected target compound (ꢁ)-18 which was converted in
two steps, via (+)-19, to (ꢁ)-7. For the purpose of com-
parative biological evaluation, diol (ꢁ)-20 was also pre-
pared by acidic hydrolysis of (ꢁ)-18 (Scheme 5).
O
NapO
HO
OH (±)-11
ONap (±)-12
Scheme 2. Reagents and conditions: (i) (CH
3
t
)
2
C(OCH
(cat.), NMO, BuOH, H
7% over two steps; (iii) PDC, DMF, rt, 2 h, 43%; (iv) DDQ, THF,
0 °C, 6 h, 20%; (v) Bu SnO, C CH , CH OH, D, then 2-bromom-
NI, C CH
, 130 °C, 9 h, 76%.
3
)
2
, p-TSA,
acetone, 0 °C to rt, 3 h; (ii) OsO
4
2
O, rt, 24 h,
5
6
2
6
H
5
3
3
The results of bioassays of compounds 7, 19 and 20 in
both racemic and enantiomerically pure forms, as well
ethylnaphthalene, Bu
4
6
H
5
3

5976
C. L. Arthurs et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5974–5977
Figure 3. Crystal structure of (±)-11 with ellipsoids at 50% probability.
HO
OCH3
OH
O
O
O
O
O
i)
ii)
iii)
OCH3
O
O
(±)-11
OCH3
OCH3
NapO
O
NapO
NapO
NapO
OCH3
OCH3
NapO
O
O
O
OH
OCH3
O
H3CO
H CO
H CO
(±)-14
(±)-15
3
3
(
±)-13
(±)-16
H CO
(±)-17
3
Scheme 3. Reagents and conditions: (i) H
CH Cl , rt, 2 h, 76%; (iii) H CO, imidazole, 1 M NaHCO
2
O:TFA:THF (5:2:1), rt, 2 h then butan-2,3-dione, (CH
(aq), THF, 40 °C, 32 h, 68%.
3 3 3
O) CH, CSA, CH OH, D, 16 h, 46%; (ii) PCC,
2
2
2
3
R
O
R
O
Table 1. Bioactivity of COTC analogues towards lung cancer cell lines
Compound
IC50 (lM)
O
O
O
O
O
O
i)
ii)
OCH3
A549
H460
(±)-17
NapO
HO
2
55
184
170
30
40
160
158
18
OCH3
(
(
(
(
(
±)-7
O
O
ꢁ)-7
H CO
H CO
3
3
(
±)-18
(±)-19
±)-19
+)-19
±)-20
60
81
36
49
iii)
iii)
R
O
R
O
O
(ꢁ)-20
41
23
O
O
Experiments were repeated three times and data within individual
experiments were derived from four separate observations: average
values are given in the table.
O
ii)
NapO
R = CH CH=CH
OH
HO
OH
OH
(±)-20
OH
centrations of each compound for 4 days and the num-
ber of surviving cells was then determined by the use of
the MTT assay.
3
(±)-7
2
1
Scheme 4. Reagents and conditions: (i) Crotonic anhydride, pyridine,
DMAP (cat.), CH Cl , rt, 3 h, 50%; (ii) DDQ, CH Cl :CH OH (4:1),
rt, 6 h, 66% for 19, 50% for 7; (iii) TFA:H O (7:1), rt, 3 h, 98% for 7,
0% for 20.
2
2
2
2
3
The results allow a number of conclusions to be reached
regarding the anti-cancer properties of the COTC ana-
logues: (i) the trihydroxylated compound, 6-epi-COTC,
in both racemic and enantiomerically pure forms, is
the least toxic of all compounds tested towards the
two cell lines; (ii) blockade of either one or two of the
free hydroxyls with a lipophilic group results in a
notable increase in potency, the extent of which is
affected to a small but measurable degree by enantio-
2
5
5
as of COMC (2), against two lung cancer cell lines
A549 and H460) are shown in Table 1. These cell lines
were chosen because they showed the highest level of
GSH from among a panel of human tumour cell lines
used in chemosensitivity testing. The cytotoxicity
assays were carried out by exposing cells to varying con-
(
2
0
R
O
R
O
I
I
R
O
OH
i), ii)
O
O
O
O
O
O
O
O
O
iii)
O
O
(-)-7
OH HO
HO
O
NapO
HO
OCH3
OCH3
2
1
OH (+)-22
NapO
OH
OH (-)-9
O
O
OH
(-)-20
H3CO
H3CO
(
-)-18
(+)-19
t
(cat.), NMO, BuOH, H
Scheme 5. Reagents and conditions: (i) (CH
3
)
2
C(OCH
3
)
, p-TSA, CH Cl
2 2
2
, rt, 1 h; (ii) OsO
4
2
O, rt, 36 h, 81% over two steps;
(
iii) H , 10% Pd on C, Et N, CH OH, rt, 3 h, 71%.
3
2
3

C. L. Arthurs et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5974–5977
5977
meric purity; (iii) all of the analogues of COTC display
slightly enhanced potency towards the chemosensitive
H460 cell line compared with the A549 cell line.
B.; Creighton, D. J. J. Med. Chem. 2003, 46, 194; (f)
Hamilton, D. S.; Zhang, X.; Ding, Z.; Hubatsch, I.;
Mannervik, B.; Houk, K. N.; Ganem, B.; Creighton, D. J.
J. Am. Chem. Soc. 2003, 125, 15049; (g) Joseph, E.;
Ganem, B.; Eiseman, J. L.; Creighton, D. J. J. Med. Chem.
It is well documented that mammalian GSTs display
broad substrate selectivity for hydrophobic compounds
and the reduced potency of 6-epi-COTC compared with
compounds 19 and 20 is consistent, therefore, with a
GST mediated mechanism for anti-cancer activity. The
observation that both mono- and di-hydroxylated ana-
logues of (1) are more potent than COMC (2) towards
lung cancer cell lines represents an interesting finding,
which is made more intriguing by small variations in
bioactivity associated with enantiomeric purity. Studies
are currently under way to further investigate the influ-
ence of absolute stereochemistry, as well as the degree of
hydroxylation of the cyclohexenone ring, on the anti-
cancer activity of this class of compounds.
2
005, 48, 6549; (h) Zheng, Z.-B.; Zhu, G.; Tak, H.; Joseph,
E.; Eiseman, J. L.; Creighton, D. J. Bioconjugate Chem.
005, 16, 598.
2
5
. Arthurs, C. L.; Wind, N. S.; Whitehead, R. C.; Stratford,
I. J. Bioorg. Med. Chem. Lett. 2007, 17, 553.
6. It should be noted that enantiomerically pure compound,
6-epi-COTC ((ꢁ)-7), possesses the same absolute stereo-
structure as the ketocarbasugar, (ꢁ)-Gabosine A: (a)
Bach, G.; Breiding-Mack, S.; Grabley, S.; Hammann, P.;
Huetter, K.; Thiericke, R.; Uhr, H.; Wink, J.; Zeeck, A.
Liebigs Ann. Chem. 1993, 241; (b) Banwell, M. G.; Bray,
A. M.; Wong, D. J. New J. Chem. 2001, 25, 1351.
. VanRheenen, V.; Kelly, R. C.; Cha, D. F. Tetrahedron
Lett. 1976, 17, 1973.
. Zamir, L. O.; Luthe, C. Can. J. Chem. 1984, 62, 1169.
. McKittrick, B. M.; Ganem, B. J. Org. Chem. 1985, 50,
5897.
7
8
9
In conclusion, expedient syntheses of the C6-diastereo-
isomer of the Streptomyces metabolite COTC, in both
racemic and enantiomerically pure forms, have been
developed using the arene-cis-dihydrodiols derived from
benzene and iodobenzene as starting materials. Bioassay
of the novel analogues, and partially protected variants
thereof, indicates that blockade of either one or two of
the free hydroxyl groups results in an increase in anti-
cancer activity. Biological potency is also affected to a
much lesser extent by the enantiomeric purity of the test
compounds. These findings indicate that further modifi-
cation of the cyclohexenone core of COTC analogues
will allow optimisation of the anti-cancer activity of this
intriguing structural class.
10. Simas, A. B. C.; Pais, K. C.; da Silva, A. A. T. J. Org.
Chem. 2003, 68, 5426.
1
1. Crystallographic data (excluding structure factors) for (±)-
1 have been deposited with the Cambridge Crystallo-
1
graphic Data Centre as supplementary publication
number CCDC 630924. Copies of the data can be
obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK [fax: +44 0
1
223336033 or e-mail: deposit@ccdc.cam.ac.uk].
12. Crystal structure data for (±)-11 C H O , M = 326.38,
20
22 4
monoclinic spacegroup C2, a = 23.915 (5), b = 5.572 (12),
˚
˚
3
3
c = 24.964 (5) A, U = 3275.9 A , dcalcd 1.323 = mgM . Inde-
pendent reflections (12,286), R1 = 0.0576, wR2 = 0.0821
for 1364 reflections with I > 2r(I).
1
3. Luo, S.; Wang, P. G.; Chen, J.-P. J. Org. Chem. 2004, 69,
5
55.
14. Basavaiah, D.; Rao, A. J.; Satynarayana, T. Chem. Rev.
003, 103, 811.
5. Rezgui, F.; El Gaied, M. M. Tetrahedron Lett. 1998, 39,
965.
Acknowledgments
2
1
1
We acknowledge, with thanks, EPSRC for funding
(
5
C.L.A. and H.L.W.) and MRC for funding (I.J.S.
6. Xia, J.; Abbas, S. A.; Locke, R. D.; Piskorz, C. F.;
Alderfer, J. L.; Matta, K. L. Tetrahedron Lett. 2000, 41,
and N.S.W). We are particularly grateful to Professor
Derek Boyd of the Queens University in Belfast for
the generous donation of diols 8 and 21 as well as for
his invaluable advice regarding the preparation of com-
pound 9 in racemic and enantiomerically pure forms.
1
69.
20
D
1
7. Selected analytical data for compound (ꢁ)-7: ½aꢂ ꢁ 15:7
ꢁ1
(c 0.28, CH OH); m
(film)/cm 3432br (O–H), 2966w
max
3
(C–H), 1647 s (C@O, enone); dH (300 MHz; D O) 1.76
2
(
4
3H, dd, J 6.9, 1.8, CH
.2, C(5)H), 4.35 (1H, d, J 10.5, C(6)H), 4.44–4.48 (1H, m,
C(4)H), 4.55–4.70 (2H, m, CH OC(@O)), 5.81 (1H, dq, J
3
CH@CH), 3.81 (1H, dd, J 10.5,
References and notes
2
1
3
5.6, 1.8, CH CH@CH), 6.90–7.03 (2H, m, C(3)H and
1
2
3
4
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CH CH@CH); d (75 MHz; D O) 19.3 (CH CH@CH),
3
C
2
3
60.3 (CH OC(@O)), 62.7 (C(5)H), 68.6 (C(6)H), 70.9
2
(C(4)H), 121.9 (CH CH@CH), C(2) not observed, 139.3
3
(C(3)H), 152.1 (CH CH@CH), 166.5 (CH OC(@O)),
3
2
192.6 (C(1)O); m/z (+ve ion electrospray) 265
265.0678,
+
([M+Na] ,100%)
(Found:
([M+Na] ) requires 265.0683).
C H O Na
11 14 6
+
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