Synthesis, reactions and structure-activity relationships of 1-hydroxyspiro[2.5]cyclooct-4-en-3-ones: Illudin analogs with in vitro cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2006.03.037

1-Hydroxyspiro[2.5]cyclooct-4-en-3-ones-analogs of natural illudines-were prepared in good yields by cyclization of 1,3-dicarbonyl dianions or 1,3-bis-silyl enol ethers ('masked dianions') with 1,1-diacylcyclopropanes. Several spirocyclopropanes showed a

Full text of DOI:10.1016/j.bmc.2006.03.037

Bioorganic & Medicinal Chemistry 14 (2006) 4694–4703
Synthesis, reactions and structure–activity relationships
of 1-hydroxyspiro[2.5]cyclooct-4-en-3-ones: Illudin analogs
with in vitro cytotoxic activity
Gopal Bose,a Karin Bracht,b Patrick J. Bednarski,b Michael Lalkb,* and Peter Langerc,d,
*
aInstitut fu¨r Chemie und Biochemie, Universita¨t Greifswald, Soldmannstr. 16, 17487 Greifswald, Germany
bInstitut fu¨r Pharmazie, Universita¨t Greifswald, Jahnstr. 17, 17487 Greifswald, Germany
cInstitut fu¨r Chemie, Universita¨t Rostock, Albert-Einstein-Str. 3a, 18059 Rostock, Germany
dLeibniz-Institut fu¨r Katalyse e. V. an der Universita¨t Rostock, Albert-Einstein-Str. 29a, 18059 Rostock, Germany
Received 19 December 2005; revised 9 March 2006; accepted 17 March 2006
Available online 17 April 2006
Abstract—1-Hydroxyspiro[2.5]cyclooct-4-en-3-ones—analogs of natural illudines—were prepared in good yields by cyclization of
1,3-dicarbonyl dianions or 1,3-bis-silyl enol ethers (‘masked dianions’) with 1,1-diacylcyclopropanes. Several spirocyclopropanes
showed a significant antiproliferative activity against human leukemia HL60 cells in vitro. 1-Hydroxyspiro[2.5]cyclooct-4-en-3-ones
represent highly reactive precursors of unstable spiro[5.2]cycloocta-4,7-dien-6-ones and reactions with a number of nucleophiles
were studied.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
for the development of new anticancer agents.3 Many
natural products with cytotoxic properties were identi-
fied as the poisonous components in fungi. One example
is the ‘jack-o-latern’ mushroom Omphalotus illudens.4
The isolation of the illudins S and M (Chart 1) as cyto-
toxic constituents of O. Illudens was reported in 1950.4
After the elucidation of their structures5—they possess
a unique 1-hydroxyspiro[5.2]cyclooct-4-en-2-one skele-
ton—several related molecules were isolated from O.
illudens and other mushrooms.6 All compounds show
cytotoxic effects in the range from very strong activity
(for Illudins S and M) to weak activity (for dehydro
and acylated derivatives).7 Spiro[2.5]cycloocta-4,7-dien-
Cytotoxic compounds play an important role as thera-
peutic agents in the treatment of cancer. After the surgi-
cal removal of a solid tumor, the systemic chemotherapy
is often used to assure a complete destruction of the
remaining tumor cells in the patients.1 Most of the che-
motherapeutic agents used today belong to alkylating
compounds (chlorambucil, melphalan, thiotepa, and
busulfan), platinum derivatives (cisplatin, carboplatin),
inhibitors of topoisomerases (camptothecin, etoposide,
and doxorubicin), antimetabolic compounds (5-fluor-
uracil, methotrexate, and hydroxyurea) or inhibitors of
mitosis (taxol, vinblastine). The latter are of special
interest because of their selective damage of specific tu-
mors in human. Like in bacterial infections tumors are
able to aquire resistance to one or more chemotherapeu-
tics.2 Therefore, the search for new cytotoxic com-
pounds is an important goal in medicinal chemistry.
Cytotoxic natural products represent lead structures
Me
Me
O
O
O
OH
Me
OEt
Me
Me
HO
Me
HO
Keywords: Cyclopropanes; Cytotoxicity; Illudins; Spiro compounds;
Structure–activity relationship.
1-Hydroxyspiro[2.5]-
cyclooct-4-en-3-one
Illudin M
*
Corresponding authors. Tel.: +49 381 4986410; fax: +49 381 4986412
(P.L.); fax: +49 3834 864885 (M.L.); e-mail addresses: lalk@
uni-greifswald.de; peter.langer@uni-rostock.de
Chart 1.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.037

G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
4695
Table 1. Products and yields
O
H
N
1
2
3
4
R1
R2
% (4)a
% (4)a
method A method B
O
H
N
MeO2C
OMe
OMe
OMe
a
b
c
a
b
c
d
b
e
a
a
a
a
b
a
a
b
c
d
e
f
OMe
OEt
Me 72
Me 70
Me 68
71
48
48
40
32
15
N
OiPr
b
b
O(CH2)2OMe Me
—
—
OEt
Me
Et
Duocarmycin SA
d
Me 58
Chart 2.
a Isolated yields.
b Experiment was not carried out.
6-ones containing aromatic rings fused to the double
bonds are present, for example, in the cytotoxic natural
products CC-1065 and duocarmycin SA (Chart 2).8
the synthesis of 4-(2-chloroethyl)salicylates by cycliza-
tion of 1,1-diacylcyclopropanes with 1,3-bis-silyl enol
ethers—masked 1,3-dicarbonyl dianions—in the pres-
ence of TiCl4 (2.0 equiv).12 Based on these results, we
developed an alternative, albeit less efficient, approach
to 1-hydroxyspiro[2.5]cyclooct-4-en-3-ones. The reac-
tion of 1,3-bis-silyl enol ether 2a, prepared from methyl
acetoacetate, afforded the 1-hydroxyspiro[5.2]cyclooct-
4-en-3-one 4a in 55% yield (Scheme 1, Table 1). During
the optimization, the use of 0.3 equiv of TiCl4 proved to
be mandatory. In fact, the use of more than 0.5 equiv of
TiCl4 resulted in cleavage of the cyclopropane moiety
and aromatisation. The employment of less than
0.3 equiv of TiCl4 resulted in a significant decrease in
yield. The cyclization of 3a with 1,3-bis-silyl enol ethers
2b–d afforded the alkoxycarbonyl-substituted 1-
hydroxyspiro[5.2]cyclooct-4-en-3-ones 4b–d. The reac-
tion of 1,1-dipropionylcyclopropane (3b) with 2b
afforded 4e. 5-Acetyl-1-hydroxyspiro[5.2]cyclooct-4-en-
3-one 4f was prepared, albeit in low yield, by cyclization
of 3a with 1,3-bis-silyl enol ether 2e prepared from
acetylacetone.
The synthesis of illudin analogs is of considerable phar-
macological relevance, due to their potential cytotoxic
and cancerostatic activity. The preparation of spi-
ro[2.5]cycloocta-4,7-dien-6-ones and their reaction with
nucleophiles, such as HBr, LiAlH4 or NaOMe, has been
previously studied.9 Padwa et al. reported an interesting
and efficient synthesis of illudins based on cyclization
reactions of diazo compounds.10 Recently, we have
reported11 the synthesis of 1-hydroxyspiro[5.2]cyclooct-
4-en-3-ones—new illudin analogs—which represent
highly reactive alkylating agents. Herein, we report full
details of the synthesis of these compounds and their
reaction with various nucleophiles. With regard to our
preliminary communication, we optimized the synthesis
of the spiro[5.2]cyclooct-4-en-3-ones (which are now
available in up to 72% yield) and studied their antipro-
liferative activity against human leukemia HL60 cells.
2. Results and discussion
2.2. Homo michael reactions of 1-hydroxyspiro[5.2]cyclo-
oct-4-en-3-ones
2.1. Synthesis of 1-hydroxyspiro[5.2]cyclooct-4-en-3-ones
Acceptor-substituted cyclopropanes represent important
building blocks in homo Michael reactions with various
nucleophiles.13,14 1-Hydroxyspiro[2.5]cyclooct-4-en-3-
ones 4 represent direct precursors to spiro[2.5]cyclooc-
ta-4,7-dien-6-ones which are formed in situ by treatment
with acid or Lewis acid and elimination of water. These
intermediates are highly reactive since they represent
vinylogous keto-substituted cyclopropanes which are
transformed into stable aromatic phenols upon cleavage
of the cyclopropane moiety. In fact, 1-hydroxyspi-
The cyclization of dilithiated methyl acetoacetate (1a)
with 1,1-diacetylcyclopropane (3a) afforded the 1-
hydroxyspiro[2.5]cyclooct-4-en-3-one 4a in up to 72%
yield. During the optimization, the stoichiometry, reac-
tion time and temperature proved to be important
parameters. The reaction of 3a with the dianions of ethyl
and isopropyl acetoacetate gave the spirocyclopropanes
4b–c in good yields. The cyclization of dilithiated
acetylacetone with 3a gave the 5-acetyl-1-hydroxyspi-
ro[5.2]cyclooct-4-en-3-one 4f. We have recently reported
O
O
OLi OLi
method A
2
2
+
R
R
1
O
O
R
R
i
1
1a-c,f
R
3a
2
R
2
HO
Me SiO OSiMe
3
3
ii
+
3a,b
1
R
4a-f
method B
2a-e
Scheme 1. Synthesis of 1-hydroxyspiro[2.5]cyclooct-4-en-3-ones; Reagents and conditions: (i) 1—LDA (2.3 equiv), 1a–c,f (1.2 equiv), THF, 1 h, 0 ꢁC,
2—3a (1.0 equiv), ꢀ78 ꢁC, 1 h, ꢀ78 to 20 ꢁC, 14 h; (ii), TiCl4 (0.3 equiv), CH2Cl2, ꢀ78 to 20 ꢁC, 12 h.

4696
G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
+
TiX
3
O
O
O
O
TiX
R
4
R
HO
Me
Me
Me
CH Cl
Me
2
2
method A
_
X
4a-c
NBu X (X = F, Cl, Br)
4
method
B
NEt X (X = I)
4
BF OEt
3
2
X
3
Ti
BF
O
3
O
O
O
R
R
Me
Me
Me
Me
_
X
+
X
NBu
4
H O
2
_
BF
3
OH O
O
O
R
H O
R
2
Me
Me
Me
Me
X
5a-h
+
X
NBu
4
Scheme 2. Reaction of 4a–c with TiX4 (method A) and NBu4X (method B).
ro[5.2]cyclooct-4-en-3-ones 4 represent highly reactive
electrophiles and strong alkylating agents.
the chloride 5d in moderate yield. The chlorination of
4b with Bu4NCl in the presence of a catalytic amount
of BF3ÆOEt2 afforded 5e in very good yield. The reaction
of 4a and 4c with TiBr4 and TiI4 afforded the halides 5f
and 5h, respectively. Likewise, the reaction of 4b and 4a
with Bu4NBr and Bu4NI afforded 5f and 5h in high
yields, respectively.
Treatment of 1-hydroxyspiro[5.2]cyclooct-4-en-3-ones 4
with titanium tetrahalides (method A) or with tetra-
alkylammonium halides in the presence of boron triflu-
oride (method B) resulted in the formation of 4-(2-
haloethyl)salicylates (Scheme 2, Table 2). The reaction
of 4c with TiF4 afforded the fluoride 5b, albeit in low
yield; the reaction of 4b with Bu4NF in the presence of
BF3OEt2 failed. Treatment of 4c with TiCl4 afforded
The reaction of 1-hydroxyspiro[5.2]cyclooct-4-en-3-ones
with acids was next studied (Scheme 3, Table 3). The
reaction of 4c with trifluoroacetic acid (TFA) afforded
the trifluoroacetate 6a. The latter was formed by acid-
mediated elimination of water to give a spiro[2.5]cyclo-
octa-4,7-dien-6-one and subsequent acid-mediated
attack of TFA onto the spirocyclopropane. Treatment
of a CH2Cl2 solution of 4b with p-toluenesulfonic acid
(TosOH) afforded the tosylate 6b. This reaction is a rare
example of a direct alkylation of p-toluenesulfonic acid
which represents a very weak nucleophile. The reaction
of 4b with glacial acetic acid (AcOH) was unsuccessful,
due to the low acidity of AcOH. However, the acetate 8c
was formed when the reaction was carried out in the
presence of BF3ÆOEt2. The reaction of a methanol solu-
Table 2. Products and yields
5
R
X
% (5)a
Method
a
b
c
d
e
f
OiPr
OEt
F
26
0
A
B
A
B
A
B
A
B
F
OiPr
OEt
Cl
Cl
Br
Br
I
53
84
94
96
47
96
OMe
OEt
OiPr
OMe
g
h
I
a Isolated yields.

G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
4697
+
The formation of 7 can be explained by BF3ÆOEt2-med-
iated elimination of water, attack of the Grignard re-
agent onto the carbonyl group, extrusion of
magnesium oxide and of a bromide ion, attack of the
latter onto the cyclopropane moiety and, finally,
aromatization.
H
O
O
O
O
R
H X
R
HO
Me
+
Me
Me
Me
+H
4a-c
2.3. Biological evaluation of 1-hydroxyspiro[5.2]cyclooct-
4-en-3-ones
H X
OH O
R
The products prepared were tested for in vitro cytotoxic
activity against the HL60 human acute promyeloid leu-
kemia cell line.15,16 Cells in the rapid phase of growth
were exposed to test compounds for 48 h. The IC50 val-
ues were determined from data of the MTT assay, which
measures cell viability.17 The results are summarized in
Table 4. For illustration, the corresponding values of
established antitumor agents as well as the IC50 values
for illudins S and M against human HL60 cells are
shown.7,18 For reasons of comparison, the cytotoxic
activity of spiro[5.4]decenones 8a–c19 (containing a
spirocyclopentane rather than a spirocyclopropane
system) and of cyclopropane 8d (containing a monocy-
clic rather than a spiro-anullated cyclopropane) was
studied (Chart 3).
_
+
H
Me
Me
X
6a-f
Scheme 3. Reaction of 4a–c with acids.
Table 3. Products and yields
6
R
X
% (6)a
a
b
c
d
e
f
OiPr
OEt
OEt
OEt
OMe
OiPr
O2C(CF3)
OAc
95
79b
61
OTos
OH
OPh
61c
57d
70d
1-Hydroxyspiro[2.5]cyclooct-4-enes 4a, 4b, 4d, and 4e
show a considerable antiproliferative activity compara-
ble in potency to carboplatin and 6-fluoruracil, and
stronger than busulfan. The activity of 4c lies in the
same range as busulfan. In contrast, spiro[5.4]decenones
8a–c and cyclopropane 8d exhibit only a weak activity.
These results show that the presence of the 1-hydroxy-
spiro[2.5]cyclooct-4-ene moiety is mandatory for cyto-
toxic activity. The presence of an ester group appears
to be important because a rather low cytotoxicity was
observed for 4f containing an acetyl rather than an ester
group. The methyl, ethyl, and methoxyethyl ester groups
of 4a, 4b, and 4d are more prone toward hydrolysis than
the isopropyl ester present in 4c which may account for
the low cell growth inhibitory activity of the latter. A
SPh
a Isolated yields.
b Carried out in the presence of BF3ÆOEt2.
c H2SO4, H2O, MeOH.
d Carried out in the presence of TFA.
tion of 4b with sulfuric acid afforded the alcohol 6d. The
replacement of BF3ÆOEt2 by TFA also gave 6d, however,
in low yield. The reaction of 4a with phenol, in the pres-
ence of TFA, afforded 6e. Likewise, the TFA-mediated
reaction of 4c with thiophenol afforded the thioether 6f.
The BF3ÆOEt2-mediated reaction of 4a with vinyl-mag-
nesium bromide afforded the styrene 7 (Scheme 4).
O
O
O
MgBr
OMe
Me
OMe
HO
Me
Me
Me
THF, CH2Cl2
BF3 OEt2
Br
7 (33%)
4a
BF3 OEt2
O
Br
MgO
O
+
OMe
Me
OMe
Me
_
MgO
Me
Me
_
Br
A
B
Scheme 4. Reaction of 4a with vinylmagnesium bromide.

4698
G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
Table 4. Results of the in vitro cytotoxic testing against human HL60
cells
3. Experimental
a
3.1. Typical procedure for the cyclization of 1,3-dicar-
bonyl dianions with 1,1-diacetylcyclopropane (method A)
Compound
IC50 (lM)
4a
4b
4.17 0.62
3.31 1.35
122 30
9.90 0.46
19.5 6.6
270b
To a THF solution (20 mL) of LDA, prepared by addi-
tion of n-BuLi (7.2 mL, 18.0 mmol, 2.5 M solution in
4c
4d
4e
8a
hexane) to
a THF solution of diisopropylamine
(2.530 g, 18.0 mmol) at 0 ꢁC. After stirring for 1 h, meth-
yl acetoacetate (1.105 g, 9.52 mmol) was added at
ꢀ78 ꢁC and the solution was stirred for 1 h. To the solu-
tion was added 1,1-diacetylcyclopropane (3a) (1.000 g,
7.9 mmol) at ꢀ78 ꢁC and the solution was allowed to
warm to 20 ꢁC during 14 h. To the reaction mixture
was added an aqueous solution of HCl (1 M) and the
organic and the aqueous layers were separated. The
aqueous layer was extracted with CH2Cl2 and the com-
bined organic layers were washed with brine, dried
(Na2SO4), filtered, and the filtrate was concentrated in
vacuo. The residue was purified by chromatography (sil-
ica gel, hexane/EtOAc) to give 4a as a colorless solid
(1.275 g, 71%).
8b
8c
119.3 17.3
140 95
437.16b
0.003 0.001c
0.003 0.001c
0.41
8d
Illudin S
Illudin M
Cisplatin
Carboplatin
Busulfan
Doxorubicin
5-Fluoruracil
6.80
61.90
0.05
16.97
a Averages and standard deviations of four independent determinations.
b (n = 1).
c Ref. 7.
3.1.1. Methyl 8-hydroxy-4,8-dimethyl-6-oxospiro[5.2]oct-
4-ene-5-carboxylate (4a). Mp = 108–109 ꢁC; Rf = 0.13
O
O
O
O
~
(hexane/EtOAc = 4:1); IR (KBr): m = 2950 (w), 1715
(s), 1669 (s), 1606 (w), 1439 (m), 1383 (m), 1248 (m),
OEt
Me
OEt
1
1160 (w) cmꢀ1; H NMR (300 MHz, CDCl3): d = 3.81
Me
HO
Me
Me
(s, 3H, OCH3), 2.72 (d, 1H, J = 15.6 Hz, CH2), 2.62
(d, 1H, J = 15.6 Hz, CH2), 2.24 (s, 1H, OH), 1.69 (s,
3H, CH3), 1.50–1.43 (m, 1H, CH2), 1.26 (s, 3H, CH3),
1.17–1.06 (m, 2H, CH2), 0.89–0.85 (m, 1H, CH2); 13C
NMR (75 MHz, CDCl3): d = 194.1, 167.6, 161.8,
132.6, 70.4 (C), 52.2 (CH3), 51.3 (CH2), 32.2 (C), 25.3,
17.0 (CH3), 11.0, 9.5 (CH2); MS (EI, 70 eV): m/z
(%) = 224 (M+, 40), 209 (30), 177 (48), 164 (69), 148
(48), 79 (30), 43 (100), 28 (37); Anal. Calcd for
C12H16O4: C, 64.27; H, 7.19. Found: C, 63.74; H, 7.71.
8b
8a
O
O
HO2C
Me
Ph
OEt
Me
O
Me
HO
HO
Me
3.1.2. 5-Acetyl-8-hydroxy-4,8-dimethyl-6-oxospiro[5.2]oct-
4-ene (4f). Starting with n-BuLi (2.74 mL, 4.3 mmol, 15%
solution in hexane), diisopropylamine (0.62 g, 4.3 mmol),
1,1-diacetylcyclopropane (3a) (252 mg, 2.0 mmol), and
acetylacetone (230 mg, 2.2 mmol) in THF (11 mL), 4f
was isolated as a yellow oil (0.242 g, 58%). IR (neat):
8d
8c
Chart 3.
~
m = 3440 (br), 2976 (m), 1700 (s), 1658 (s), 1597 (s), 1382
(s), 1354 (s), 1280 (m), 1160 (m), 1106 (m), 1067 (m)
cmꢀ1; H NMR (300 MHz, CDCl3): d = 3.10 (br, 1H,
1
very strong antiproliferative activity has been reported
for illudins S and M.7 It has been established that the
compounds react as alkylating agents, following the
reaction of a nucleophile (such as glutathione) with the
unsaturated ketone moiety. The resultant cyclohexadi-
ene intermediate rapidly aromatizes, with concurrent
ring opening of the cyclopropane ring, which can trap
DNA. The generation of such a cyclohexadiene interme-
diate is more difficult for 4a–e, which is presumably the
reason why these compounds are less active than the
illudins. In addition, an important difference between
4a–e and the illudins lies in the fact that the latter are
double alkylation agents.20 In conclusion, the cytotoxic-
ities of readily accessible spirocyclopropanes 4a, b, d, e
are—albeit lower than those of the rather complex nat-
ural products—very promising.
OH), 2.73 (d, 1H, J = 15.6 Hz, CH2), 2.59 (d, 1H,
J = 15.6 Hz, CH2), 2.32 (s, 3H, COCH3), 1.63 (s, 3H,
CH3), 1.50–1.40 (m, 1H, CH2), 1.25 (s, 3H, CH3), 1.15–
1.06 (m, 2H, CH2), 0.82–0.88 (m, 1H, CH2); 13C NMR
(75 MHz, CDCl3): d = 205.0 (COCH3), 196.1 (CO),
161.4, 139.0 (C), 70.1 (COH), 51.6 (CH2), 32.4 (C, spiro),
31.6, 24.9, 16.1 (CH3), 11.0, 9.5 (CH2); MS (EI, 70 eV):
m/z (%) = 208 (M+, 10), 193 (24), 175 (8), 165 (21).
3.2. Typical procedure for the cyclization of 1,3-bis-silyl
enol ethers with 1,1-diacylcyclopropanes (method B)
To a CH2Cl2 solution (100 mL) of 1,1-diacetylcyclopro-
pane (3a) (0.190 g, 1.5 mmol) and of 1-methoxy-1,3-
bis(trimethylsilyloxy)buta-1,3-diene
(2a)
(0.580 g,

G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
4699
˚
2.2 mmol) in the presence of molecular sieves (4 A,
1.0 g) was dropwise added a CH2Cl2 solution (1 mL)
of TiCl4 (0.05 mL 0.45 mmol) at ꢀ78 ꢁC under argon
atmosphere. The reaction mixture was allowed to warm
to 20 ꢁC over 6 h and was stirred for additional 6 h at
20 ꢁC. The solution was filtered and the filtrate was
poured into an aqueous solution of HCl (1.0 M,
100 mL). The organic and the aqueous layer were sepa-
rated and the latter was extracted with CH2Cl2 (3·
100 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated in vacuo. The resi-
due was purified by column chromatography (silica gel,
hexane/EtOAc = 4:1 ! 1:1) to give 4a (0.244 g, 72%) as
a colorless solid (for spectroscopic data, see above).
3.2.3. Ethyl 8-hydroxy-4,8-diethyl-6-oxospiro[5.2]oct-4-
ene-5-carboxylate (4e). The reaction was carried out
according to the procedure as given for the synthesis of
4a. Starting with 1,1-dipropionylcyclopropane (3b)
(0.154 g, 1.0 mmol), 1-ethoxy-1,3-bis(trimethylsilyl-
oxy)buta-1,3-diene (2b) (0.410 g, 1.5 mmol) and TiCl4
(0.035 mL in 1 mL CH2Cl2, 0.3 mmol), 4e was isolated
as a yellow oil (0.085 g, 32%); Rf = 0.18 (hexane/ethyl ace-
1
tate = 1:1); H NMR (300 MHz, CDCl3): d = 4.15–4.40
(m, 2H, OCH2), 2.72 (d, 1H, J = 15.6 Hz, CH2), 2.65 (d,
1H, J = 15.6 Hz, CH2), 2.10–2.25 (m, 1H, CH2), 1.70–
1.85 (m, 1H, CH2), 1.50–1.65 (m, 2H, CH2), 1.42–1.50
(m, 1H, CH2), 1.32 (t, 3H, J = 7.0 Hz, CH3), 1.15–1.25
(m, 1H, CH2), 1.05–1.12 (m, 1H, CH2), 1.10 (t, 3H,
J = 7.0 Hz, CH3), 0.92 (t, 3H, J = 7.0 Hz, CH3), 0.60–
0.70 (m, 1H, CH2); 13C NMR (75 MHz, CDCl3):
d = 194.2, 167.1, 166.3, 132.5, 72.1 (C), 61.3, 48.8 (CH2),
30.3 (C), 29.6, 23.9 (CH2), 14.2, 13.2 (CH3), 10.5, 8.9
(CH2), 7.4 (CH3); MS (EI, 70 eV): m/z (%) = 266 (M+,
12), 237 (25), 221 (20), 181 (85).
3.2.1. Ethyl 8-hydroxy-4,8-dimethyl-6-oxospiro[5.2]oct-4-
ene-5-carboxylate (4b). The reaction was carried out
according to the procedure as given for the synthesis of
4a. Starting with 1,1-diacetylcyclopropane (3a) (0.200 g,
1.6 mmol), 1-ethoxy-1,3-bis(trimethylsilanyloxy)buta-
1,3-diene (2b) (0.650 g, 2.4 mmol), and TiCl4 (0.05 mL
in 1 mL CH2Cl2, 0.5 mmol), 4b was isolated as a colorless
solid (0.180 g, 48%), mp = 56–57 ꢁC; Rf = 0.15 (hexane/
3.3. Typical procedure for the reaction of 1-hydroxyspi-
ro[2.5]cyclooct-4-en-3-ones with titanium tetrahalides
(method A)
~
ethyl acetate = 4:1); IR (KBr): m = 3483 (m), 2980 (m),
1730 (s), 1666 (s), 1606 (m), 1439 (m), 1383 (m), 1248
;
(m), 1170 (w) cmꢀ1
1H NMR (300 MHz, CDCl3):
To a CH2Cl2 solution (0.5 mL) of isopropyl-8-hydroxy-
4,8-dimethyl-6-oxospiro[2.5]oct-4-ene-5-carboxylate (4c)
(0.035 g, 0.1 mmol) was dropwise added TiF4 (0.017 g,
0.14 mmol) at ꢀ78 ꢁC under argon atmosphere. The
reaction mixture was allowed to warm to 20 ꢁC over
6 h and was stirred for additional 6 h at 20 ꢁC. The solu-
tion was filtered and the filtrate was poured into an
aqueous solution of HCl (1.0 M, 100 mL). The organic
and the aqueous layer were separated and the latter
was extracted with CH2Cl2 (3· 100 mL). The combined
organic layers were dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by
column chromatography (silica gel, hexane/EtOAc =
4:1 ! 1:1) to give 5a (0.009 g, 26%) as a colorless oil
(for spectroscopic data, see below).
d = 4.29 (d, 2H, J = 7.2 Hz, OCH2), 2.72 (d, 1H,
J = 15.6 Hz, CH2), 2.62 (d, 1H, J = 15.6 Hz, CH2), 2.06
(s, 1H, OH), 1.69 (s, 3H, CH3), 1.49–1.42 (m, 1H, CH2),
1.32 (t, 3H, J = 7.2 Hz, CH3), 1.27 (s, 3H, CH3), 1.16–
1.06 (m, 2H, CH2), 0.89–0.82 (m, 1H, CH2); 13C NMR
(75 MHz, CDCl3): d = 194.2, 167.2, 161.4, 132.7, 70.3
(C), 61.3, 51.3 (CH2), 32.1 (C), 25.2, 16.8, 14.8 (CH3),
10.9, 9.4 (CH2); MS (EI, 70 eV): m/z (%) = 238 (M+,
28), 223 (33), 193 (22), 177 (34), 164 (44), 149 (69), 43
(100); HRMS (EI, 70 eV): calcd for C13H18O4: m/
z = 238.1205; found: m/z = 238.1205 2 mD (M+); Anal.
Calcd for C13H18O4: C, 65.52; H, 7.61. Found: C, 65.98;
H, 7.52.
3.2.2.
Isopropyl
8-hydroxy-4,8-dimethyl-6-oxospi-
ro[5.2]oct-4-ene-5-carboxylate (4c). The reaction was
carried out according to the procedure as given for the
synthesis of 4a. Starting with 1,1-diacetylcyclopropane
(3a) (0.131 g, 1.0 mmol), 1-isopropyloxy-1,3-bis(trimeth-
ylsilyloxy)buta-1,3-diene (2c) (0.450 g, 1.6 mmol) and
TiCl4 (0.03 mL in 1 mL CH2Cl2, 0.3 mmol), 4c was iso-
lated as a colorless oil (0.125 g, 48%); Rf = 0.18 (hexane/
3.3.1. Isopropyl 3-(2-fluoroethyl)-6-hydroxy-2,4-dimethyl-
benzoate (5a). Starting with isopropyl-8-hydroxy-4,8-di-
methyl-6-oxospiro[2.5]oct-4-ene-5-carboxylate (4c) (0.035 g,
0.14 mmol), TiF4 (0.017 g, 0.14 mmol), and CH2Cl2
(0.5 mL), 5a (0.009 g, 26%) was isolated as a colorless oil;
~
Rf = 0.67 (hexane/EtOAc = 9:1); IR (neat): m = 2979 (m),
1720 (w), 1655 (s), 1603 (m), 1574 (m), 1467 (s), 1370 (s),
1
ethyl acetate = 1:1); IR (neat): m = 3399 (br), 2983 (w),
1244 (s), 1105 (s), 804 (w) cmꢀ1; H NMR (300 MHz,
~
1729 (s), 1659 (s), 1617 (m), 1380 (m), 1243 (s), 1024
CDCl3): d = 10.68 (s, 1H, OH), 6.68 (s, 1H, ArH), 5.31
(sep, 1H, J = 6.2 Hz, OCH), 3.45 (t, 2H, J= 7.5 Hz,
CH2Cl), 2.92 (t, 2H, J = 7.5 Hz, CH2), 2.49 (s, 3H, CH3),
2.32 (s, 3H, CH3), 1.39 (d, 6H, J = 6.3 Hz, CH3); 13C
NMR (75 MHz, CDCl3): d = 170.9, 159.7, 144.2, 138.9,
127.2 (C), 116.9 (CH), 112.3 (C), 69.8 (CH2), 69.6 (CH),
29.8 (CH2), 21.9 (C), 21.1, 18.7 (CH3); elemental analysis:
calcd (%) for C14H19O3F: C, 66.12; H, 7.53; found: C,
66.51; H, 7.26.
(m), 745 (w) cmꢀ1 1H NMR (300 MHz, CDCl3):
;
d = 5.17 (sept, 1H, J = 6.3 Hz, OCH), 2.71 (d, 1H,
J = 15.6 Hz, CH2), 2.59 (d, 1H, J = 15.6 Hz, CH2),
2.40 (br, 1H, OH), 1.68 (s, 3H, CH3), 1.49–1.37 (m,
1H, CH2), 1.30 (d, 6H, J = 6.3 Hz, CH3), 1.26 (s, 3H,
CH3), 1.15–1.04 (m, 2H, CH2), 0.88–0.81 (m, 1H,
CH2); 13C NMR (75 MHz, CDCl3): d = 194.0, 166.8,
160.5, 133.2, 70.5 (C), 69.0 (CH), 51.5 (CH2), 32.1 (C),
25.4, 21.7 (2C), 16.7 (CH3), 10.8, 9.5 (CH2); MS (EI,
70 eV): m/z (%) = 252 (M+, 40), 237 (13), 193 (65), 177
(41), 164 (47), 148 (100), 91 (17), 43 (78); HRMS
(EI, 70 eV): calcd for C14H20O4: m/z = 252.1362; found:
m/z = 252.1362 2 mD.
3.3.2. Isopropyl 3-(2-chloroethyl)-6-hydroxy-2,4-dimethyl-
benzoate (5c). Starting with isopropyl 8-hydroxy-4,8-di-
methyl-6-oxospiro[2.5]oct-4-ene-5-carboxylate (4c) (0.050 g,
0.20 mmol), TiCl4 (0.014 mL, 0.14 mmol; dissolved in

4700
G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
1 mL of CH2Cl2), CH2Cl2 (3 mL), and molecular sieves
˚
(4 A), 5c was isolated (0.029 g, 53%) as a colorless solid,
dropwise added BF3ÆOEt2 (0.01 mL, 0.07 mmol; dis-
solved in 0.2 mL of CH2Cl2) at ꢀ78 ꢁC under argon atmo-
sphere. The reaction mixture was allowed to warm to
20 ꢁC over 6 h and was stirred for additional 6 h at
20 ꢁC. The solution was filtered and the filtrate was
poured into an aqueous solution of HCl (1.0 M,
100 mL). The organic and the aqueous layers were sepa-
rated and the latter was extracted with CH2Cl2 (3·
100 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated in vacuo. The resi-
due was purified by column chromatography (silica gel,
hexane/EtOAc = 4:1 ! 1:1) to give 5d (0.028 g, 84%) as
a colorless solid (for spectroscopic data, see below).
mp = 51–52 ꢁC; Rf = 0.68 (hexane/EtOAc = 4:1); IR
~
(KBr): m = 2982 (m), 1731 (w), 1656 (s), 1601 (w), 1574
(m), 1467 (m), 1372 (s), 1238 (s), 1105 (m), 804 (w), 703
(w) cmꢀ1; 1H NMR (300 MHz, CDCl3): d = 10.77 (s, 1H,
OH), 6.70 (s, 1H, ArH), 5.32 (sep, 1H, J = 6.2 Hz, OCH),
3.53–3.46 (m, 2H, CH2), 3.12–3.06 (m, 2H, CH2), 2.50 (s,
3H, CH3), 2.33 (s, 3H, CH3), 1.46 (d, 6H, J = 6.2 Hz,
CH3); 13C NMR (75 MHz, CDCl3): d = 170.8, 160.2,
143.9, 138.9, 127.0 (C), 117.2 (CH), 112.3 (C), 69.7
(OCH), 42.2, 33.0 (CH2), 21.9 (C), 21.0, 18.6 (CH3); MS
(EI, 70 eV): m/z (%) = 272 (M++2, 5), 270 (M+, 15), 212
(26), 210 (74), 161 (100), 91 (8), 77 (7), 28 (35); elemental
analysis: calcd (%) for C14H19O3Cl: C, 62.10; H, 7.07;
found: C, 62.07; H, 7.49.
3.4.1. Ethyl 3-(2-chloroethyl)-6-hydroxy-2,4-dimethyl-
benzoate (5d). Starting with ethyl 8-hydroxy-4,8-dimeth-
yl-6-oxospiro[2.5]oct-4-ene-5-carboxylate (4b) (0.031 g,
0.13 mmol), n-Bu4NCl (0.037 g, 0.13 mmol), CH2Cl2
(1 mL), and BF3ÆOEt2 (0.01 mL, 0.07 mmol; dissolved in
0.2 mL of CH2Cl2), 5d was isolated (0.028 g, 84%) as a col-
orless solid, mp = 53–54 ꢁC; Rf = 0.64 (hexane/EtOAc =
3.3.3. Methyl 3-(2-bromoethyl)-6-hydroxy-2,4-dimethyl-
benzoate (5e). Starting with methyl 8-hydroxy-4,8-dimeth-
yl-6-oxospiro[2.5]oct-4-ene-5-carboxylate (4a) (0.029 g,
0.20 mmol), TiBr4 (0.048 mL, 0.13 mmol), and CH2Cl2
(1 mL), 5e was isolated (0.035 g, 94%) as a colorless solid,
mp = 73–74 ꢁC; Rf = 0.63 (hexane/EtOAc = 4:1); IR
~
4:1); IR (KBr): m = 2978 (m), 1666 (s), 1606 (w), 1561
(m), 1467 (m), 1311 (s), 1072 (m), 699 (w) cmꢀ1 1H
;
~
(KBr): m = 2950 (m), 1721 (w), 1656 (s), 1599 (m), 1574
NMR (300 MHz, CDCl3): d = 10.74 (s, 1H, OH), 6.70 (s,
1H, ArH), 4.43 (q, 2H, J = 7.2 Hz, OCH2), 3.52–3.47 (m,
2H, CH2), 3.13–3.07 (m, 2H, CH2), 2.51 (s, 3H, CH3),
2.34 (s, 3H, CH3), 1.42 (t, 3H, J = 7.2 Hz, CH3); 13C
NMR (75 MHz, CDCl3): d = 171.3, 160.3, 144.0, 139.0,
127.0 (C), 117.2 (CH), 112.0 (C), 61.6, 42.2, 32.9 (CH2),
21.0, 18.6, 14.1 (CH3); MS (EI, 70 eV): m/z (%) = 258.4
(M++2, 12), 256 (M+, 38), 212 (34), 210 (88), 161 (100),
91(5), 77(4);elemental analysis: calcd (%) for C13H17O3Cl:
C, 60.82; H, 6.67; found: C, 60.81; H, 7.09.
(m), 1436 (s), 1355 (s), 1237 (s), 1071 (m), 805 (w) cmꢀ1
;
1H NMR (300 MHz, CDCl3): d = 10.69 (s, 1H, OH),
6.69 (s, 1H, ArH), 3.95 (s, 3H, OCH3), 3.35–3.29 (m, 2H,
CH2), 3.19–3.15 (m, 2H, CH2), 2.47 (s, 3H, CH3), 2.32 (s,
3H, CH3); 13C NMR (75 MHz, CDCl3): d = 171.8, 160.3,
144.0, 138.8, 128.2 (C), 117.3 (CH), 111.8 (C), 52.2
(OCH3), 33.3, 29.7 (CH2), 20.9, 18.5 (CH3); MS (EI,
70 eV): m/z (%) = 288 (M++2, 24), 286 (M+, 26), 256
(63), 254 (62), 207 (62), 193 (23), 175 (31), 161 (100), 77
(12); elemental analysis: calcd (%) for C12H15O3Br: C,
50.13; H, 5.26; found: C, 50.29; H, 5.43.
3.4.2. Ethyl 3-(2-bromoethyl)-6-hydroxy-2,4-dimethyl-
benzoate (5f). Starting with ethyl 8-hydroxy-4,8-dimethyl-
6-oxospiro[2.5]oct-4-ene-5-carboxylate (4b) (0.030 g,
0.13 mmol) and n-Bu4NBr (0.048 g, 0.15 mmol), CH2Cl2
(1 mL), and BF3ÆOEt2 (0.01 mL, 0.07 mmol; dissolved in
0.2 mL of CH2Cl2), 5f was isolated (0.037 g, 95%) as a col-
orless solid, mp = 49–50 ꢁC; Rf = 0.61 (hexane/EtOAc =
3.3.4. Isopropyl 6-hydroxy-3-(2-iodoethyl)-2,4-dimethyl-
benzoate (5g). Starting with isopropyl 8-hydroxy-4,8-di-
methyl-6-oxospiro[2.5]oct-4-ene-5-carboxylate (4c) (0.025 g,
0.10 mmol), TiI4 (0.054 mL, 0.10 mmol), and CH2Cl2
(1 mL), 5g was isolated (0.018 g, 53%) as a colorless oil;
~
~
Rf = 0.77 (hexane/EtOAc = 4:1); IR (neat): m = 2980 (w),
1657 (s), 1603 (w), 1574 (w), 1464 (m), 1369 (s), 1236 (s),
4:1); IR (KBr): m = 2978 (m), 1666 (s), 1606 (w), 1561
(m), 1467 (m), 1311 (s), 1072 (m), 699 (w) cmꢀ1 1H
;
1
1103 (m), 803 (m) cmꢀ1; H NMR (300 MHz, CDCl3):
NMR (300 MHz, CDCl3): d = 10.76 (s, 1 H, OH), 6.69
(s, 1H, ArH), 4.43 (q, 2H, J = 7.2 Hz, OCH2), 3.36–3.30
(m, 2H, CH2), 3.19–3.13 (m, 2H, CH2), 2.50 (s, 3H,
CH3), 2.32 (s, 3H, CH3), 1.42 (t, 3H, J = 7.2 Hz, CH3);
13C NMR (75 MHz, CDCl3): d = 171.3, 160.3, 143.9,
138.9, 128.2 (C), 117.3 (CH), 112.0 (C), 61.6, 33.4, 29.7
(CH2), 20.9, 18.5, 14.1 (CH3); MS (EI, 70 eV): m/z
(%) = 303 (M++2, 11), 300 (M+, 11), 256 (32), 254 (32),
221 (20), 175 (29), 161 (100), 91 (23), 77 (18), 29 (31); ele-
mental analysis: calcd (%) for C13H17O3Br: C, 60.82; H,
6.67; found: C, 60.81; H 7.09.
d = 10.79 (s, 1H, OH), 6.69 (s, 1H, ArH), 5.32 (sep, 1H,
J = 6.2 Hz, OCH), 3.19–3.15 (m, 2H, CH2I), 3.12–3.06
(m, 2H, CH2), 2.47 (s, 3H, CH3), 2.30 (s, 3H, CH3), 1.40
(d, 6H, J = 6.2 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d = 170.8, 160.2, 143.4, 138.5, 130.6 (C), 117.3 (CH),
112.4 (C), 69.8 (CH2), 34.8 (CH), 21.9 (2C), 20.9, 18.6
(CH3), 1.8 (CH2); MS (EI, 70 eV): m/z (%) = 362 (M+,
11), 303 (14), 235 (61), 175 (100), 161 (14), 91 (10), 28
(10); the exact molecular mass for C14H19O3I m/
z = 362.0379 2 mD (M+) was confirmed by HRMS (EI,
70 eV).
3.4.3. Methyl 6-hydroxy-3-(2-iodoethyl)-2,4-dimethylben-
zoate (5h). Starting with methyl 8-hydroxy-4,8-dimethyl-6-
oxospiro[2.5]oct-4-ene-5-carboxylate (4a) (0.032 g, 0.14
mmol), Et4NI (0.036 g, 0.14 mmol), CH2Cl2 (1 mL), and
BF3ÆOEt2 (0.01 mL, 0.07 mmol; dissolved in 0.2 mL of
CH2Cl2), 5hwas isolated(0.046 g, 96%) asa colorless solid,
mp = 94–95 ꢁC; Rf = 0.62 (hexane/EtOAc = 4:1); IR
3.4. Typical procedure for the reaction of 1-hydroxyspi-
ro[2.5]cyclooct-4-en-3-ones with tetraalkylammonium
halides (method B)
ToaCH2Cl2 solution (1 mL)ofethyl8-hydroxy-4,8-dimeth-
yl-6-oxospiro[2.5]oct-4-ene-5-carboxylate (4b) (0.031 g,
0.13 mmol) and of n-Bu4NCl (0.037 g, 0.13 mmol) was
~
(neat): m = 2980 (w), 1657 (s), 1603 (w), 1574 (w), 1464

G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
4701
(m), 1369 (s), 1236 (s), 1103 (m), 803 (m) cmꢀ1; 1H NMR
(300 MHz, CDCl3): d = 10.73 (s, 1H, OH), 6.70 (s, 1H,
ArH), 3.95 (s, 3H, OCH3), 3.22–3.15 (m, 2H, CH2I),
3.12–3.05 (m, 2H, CH2), 2.47 (s, 3H, CH3), 2.30 (s, 3H,
CH3), 1.40 (d, 6H, J = 6.2 Hz, CH3); 13C NMR
(75 MHz, CDCl3): d = 171.8, 160.2, 143.7, 138.5, 130.7
(C), 117.3 (CH), 111.9 (C), 52.2 (CH3), 34.7 (CH2), 20.9,
18.5 (CH3), 1.7 (CH2); MS (EI, 70 eV): m/z (%) = 334
(M+, 9), 303 (4), 207 (79), 175 (100), 161 (24), 147 (17),
119 (18), 91 (25), 77 (17), 28 (24); elemental analysis calcd
(%) for C12H15O3I: C, 43.13; H, 4.52; found: C, 42.81; H,
4.37.
CH3), 2.37 (s, 3H, CH3), 2.21 (s, 3H, CH3), 1.41 (t,
3H, J = 7.2 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d = 171.2, 160.3, 144.7, 144.2, 139.1, 132.9 (C), 129.7
(C), 127.6 (CH), 124.7 (C), 117.2 (CH), 112.0 (C),
68.5, 61.6, 28.8 (CH2), 20.9, 20.5, 18.4, 14.1 (CH3);
MS (EI, 70 eV): m/z (%) = 392 (M+, 34), 346 (82), 207
(25), 174 (82), 161 (100), 91 (35), 28 (57).
3.4.6. Ethyl 3-(2-acetoxyethyl)-6-hydroxy-2,4-dimethyl-
benzoate (6c). The reaction was carried out following the
procedure as given for the synthesis of 6b. The BF3ÆOEt2-
was added last to the reaction mixture. Starting with ethyl
8-hydroxy-4,8-dimethyl-6-oxospiro[2.5]oct-4-ene-5-carbox-
ylate (4b) (0.040 g, 0.17 mmol), glacial acetic acid (0.02 mL,
0.34 mmol), CH2Cl2 (1 mL), and BF3ÆOEt2 (0.01 mL,
0.07 mmol), 6c was isolated (0.038 g, 79%) as a colorless
3.4.4. Isopropyl 6-hydroxy-2,4-dimethyl-3-[2-(2,2,2-triflu-
TFA
oroacetoxy)ethyl]benzoate
(6a).
(0.04 mL,
0.5 mmol) was added dropwise at 20 ꢁC to a CH2Cl2
solution (1.0 mL) of isopropyl 8-hydroxy-4,8-dimethyl-
6-oxospiro[5.2]oct-4-ene-5-carboxylate (4c) (0.064 g,
0.3 mmol) and the reaction mixture was stirred for 4 h
(monitored by TLC). The solvent and TFA were re-
moved in vacuo and the residue was purified by column
chromatography (silica gel, hexane/EtOAc = 19:1) to
give 6a (0.084 g, 95%) as a colorless solid; mp = 32–
33 ꢁC; Rf = 0.58 (hexane/EtOAc = 19:1); IR (KBr):
~
oil; Rf = 0.53 (hexane/EtOAc = 4:1); IR (neat): m = 3420
(w), 2980 (m), 1739 (s), 1658 (s), 1605 (m), 1574 (w), 1467
(m), 1372 (m), 1244 (s), 1041 (m), 804 (w) cmꢀ1; 1H NMR
(300 MHz, CDCl3): d = 10.70 (s, 1H, OH), 6.70 (s, 1H,
ArH), 4.42 (q, 2H, J = 7.2 Hz, OCH2), 4.09 (t, 2H,
J = 7.8 Hz, OCH2), 2.96 (t, 2H, J = 7.8 Hz, CH2), 2.53 (s,
3H, CH3), 2.35 (s, 3H, CH3), 2.06 (s, 3H, CH3), 1.42 (t,
3H, J = 7.2 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d = 171.4, 171.0, 160.6, 144.4, 139.2, 126.2 (C), 117.1 (CH),
112.0 (C), 62.9, 61.5, 28.5 (CH2), 21.0, 21.0, 18.6, 14.2
(CH3); MS (EI, 70 eV): m/z (%) = 288 (M+, 19), 235 (11),
220 (31), 174 (100), 161 (83), 41 (17); elemental analysis: calcd
(%) for C15H20O5: C, 64.27; H, 7.19; found: C, 64.25; H, 7.59.
~
m = 2959 (s), 1786 (m), 1657 (s), 1453 (s), 1375 (s),
1
1166 (s), 1105 (m) cmꢀ1; H NMR (300 MHz, CDCl3):
d = 10.77 (s, 1H, OH), 6.71 (s, 1H, ArH), 5.32 (sep,
1H, J = 6.3 Hz, OCH), 4.37 (t, 2H, J = 8.1 Hz, CH2F),
3.08 (t, 2H, J = 8.1 Hz, CH2), 2.53 (s, 3H, CH3), 2.35
(s, 3H, CH3), 1.40 (d, 3H, J = 6.3 Hz, CH3), 1.39 (d,
3H, J = 6.3 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d = 170.8 (2C), 160.3, 157.5 (q, J = 42.1 Hz, CF3),
144.1, 139.3, 124.5 (C), 117.3 (CH), 112.5 (C), 69.8
(CH), 66.3, 27.9 (CH2), 21.9 (C), 20.9, 18.6 (CH3); MS
(EI, 70 eV): m/z (%) = 348 (M+, 68), 306 (41), 288
(100), 161 (93), 91 (31), 43 (34); the exact molecular mass
for C16H19O5F3 m/z = 348.1185 2 mD (M+) was con-
firmed by HRMS (EI, 70 eV).
3.4.7. Ethyl 6-hydroxy-3-(2-hydroxyethyl)-2,4-dimethyl-
benzoate (6d). The reaction was carried out following the
procedure as given for the synthesis of 6b. Acetone rather
than CH2Cl2 was used for the reaction. Starting with ethyl
8-hydroxy-4,8-dimethyl-6-oxospiro[2.5]oct-4-ene-5-carbox-
ylate (4b) (0.045 g, 0.19 mmol), sulfuric acid (10%, 0.5 mL),
and acetone (0.5 mL), 6d was isolated (0.017 g, 39%) as a col-
orless oil; Rf = 0.13 (hexane/EtOAc = 4:1); IR (KBr):
~
m = 3339 (br), 2958 (m), 1722 (s), 1660 (s), 1603 (m), 1575
(w), 1442 (s), 1350 (m), 1244 (s), 1040 (s), 856 (w), 805 (w)
3.4.5. Ethyl 6-hydroxy-2,4-dimethyl-3-[2-(toluene-4-sulfo-
nyloxy)ethyl]benzoate (6b). To a CH2Cl2 solution (1 mL)
of ethyl 8-hydroxy-4,8-dimethyl-6-oxospiro[2.5]oct-4-
ene-5-carboxylate (4b) (0.045 g, 0.19 mmol) was added
a CH2Cl2 solution of PTSA (0.036 g, 0.19 mmol) at
ꢀ78 ꢁC under argon atmosphere. The reaction mixture
was allowed to warm to 20 ꢁC over 6 h and was stirred
for additional 6 h at 20 ꢁC. The solution was filtered
and the filtrate was poured into an aqueous solution
of HCl (1.0 M, 100 mL). The organic and the aqueous
layer were separated and the latter was extracted with
CH2Cl2 (3· 100 mL). The combined organic layers were
dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by column chromatography (silica
gel, hexane/EtOAc = 4:1 ! 1:1) to give 6b (0.045 g,
53%) as a colorless oil which solidified upon standing;
mp = 73–74 ꢁC; Rf = 0.35 (hexane/EtOAc = 4:1); IR
cmꢀ1 1H NMR (300 MHz, CDCl3): d = 10.66 (s, 1H,
;
OH), 6.70 (s, 1H, ArH), 4.42 (q, 2H, J = 7.2 Hz, OCH2),
3.71 (t, 2H, J = 7.5 Hz, OCH2), 2.93 (t, 2H, J = 7.5 Hz,
CH2), 2.51 (s, 3H, CH3), 2.34 (s, 3H, CH3), 1.44 (t, 3H,
J = 7.2 Hz, CH3); 13C NMR (75 MHz, CDCl3): d = 171.5,
159.8, 144.4, 139.0, 127.0 (C), 117.0 (CH), 112.0 (C), 61.8,
61.5, 32.4 (CH2), 21.2, 18.7, 14.2 (CH3); MS (EI, 70 eV):
m/z (%) = 238 (M+, 66), 207 (69), 192 (71), 161 (100), 104
(25), 79 (19); the exact molecular mass for C13H18O4: m/
z = 238.1164 2 mD was confirmed by HRMS (EI, 70 eV).
3.4.8. Methyl 6-hydroxy-2,4-dimethyl-3-(2-phenoxyeth-
yl)benzoate (6e). The reaction was carried out following
the procedure as given for the synthesis of 6a. Starting
with methyl 8-hydroxy-4,8-dimethyl-6-oxospiro[2.5]oct-
4-ene-5-carboxylate (4a) (0.045 g, 0.20 mmol), phenol
(0.025 g, 0.27 mmol), CH2Cl2 (1 mL), and TFA (two
drops), 6e was isolated (0.038 g, 79%) as a colorless sol-
id, mp = 78–79 ꢁC; Rf = 0.53 (hexane/EtOAc = 4:1); IR
~
(neat): m = 3432 (br), 1717 (w), 1654 (s), 1602 (m),
1574 (w), 1468 (m), 1351 (s), 1240 (s), 1179 (s), 965 (w)
1
cmꢀ1; H NMR (300 MHz, CDCl3): d = 10.71 (s, 1 H,
~
OH), 7.67 (dd, 2H, J= 2.1 Hz, 8.1 Hz, ArH), 7.27 (dd,
2H, J = 2.1 Hz, 8.1 Hz, ArH), 6.62 (s, 1H, ArH), 4.41
(q, 2H, J = 7.2 Hz, OCH2), 4.04 (t, 2H, J = 7.8 Hz,
OCH2), 2.99 (t, 2H, J = 7.8 Hz, CH2), 2.42 (s, 3H,
(neat): m = 3398 (w), 2980 (m), 1738 (s), 1658 (s), 1605
(m), 1574 (w), 1465 (s), 1375 (m), 1244 (s), 1035 (s),
1
863 (w), 804 (w) cmꢀ1; H NMR (300 MHz, CDCl3):
d = 10.64 (s, 1H, OH), 7.29–7.24 (m, 2H, ArH), 6.96–

4702
G. Bose et al. / Bioorg. Med. Chem. 14 (2006) 4694–4703
6.86 (m, 3H, ArH), 6.72 (s, 1 H, ArH), 3.97 (t, 2H,
J = 7.2 Hz, OCH2), 3.95 (s, 3H, OCH3), 3.14 (t, 2H,
J = 7.5 Hz, CH2), 2.53 (s, 3H, CH3), 2.37 (s, 3H,
CH3); 13C NMR (75 MHz, CDCl3): d = 172.1, 160.2,
159.0, 144.8, 139.4 (C), 129.7 (CH), 127.1 (C), 121.0,
17.4, 114.7 (CH), 112.2 (C), 66.8 (CH2), 52.3 (CH3),
29.6 (CH2), 21.4, 18.9 (CH3); MS (EI, 70 eV): m/z
(%) = 300 (M+, 11), 269 (3), 207 (3), 193 (49), 175 (4),
161 (100), 133 (5), 105 (8), 77 (9), 28 (28); the exact
molecular mass for C18H20O4: m/z = 300.1362 2 mD
was confirmed by HRMS (EI, 70 eV); elemental analy-
sis: calcd (%) for C18H20O4: C, 71.98; H, 6.71; found:
C, 71.67; H, 7.09.
3.5. Cytotoxicity assay
All chemicals were purchased from SIGMA and used
without further purification. A human leukemia cell line
HL60 (acute promyeloid leukemia, FAB-subtype M2)
was used for the antitumor assay investigations (source:
DSMZ, Braunschweig, Germany, No. ACC3). The
growth medium consists of RPMI1640 medium includ-
ing 10% FCS, 15 mg of penicillin and 27 mg of gentami-
cin-sulfate per 500 mL. The cells were incubated for 24 h
at 37 ꢁC, 5% CO2, 95% humidity as a discrete cell sus-
pension.15,16 The final concentration was 100,000 cells/
mL. The test compounds were dissolved in DMSO
and serially diluted to five concentrations for testing
(end concentration 1:1000 in medium). Negative con-
trols were performed with just DMSO. The MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide)
assay was performed according to a known procedure17 with
2.5 mg of MTT per 1 mL of PBS (including 0.15 M NaCl
and 0.02 M sodium phosphate); 20 lL was added per well.
After an incubation time of 48 h at 37 ꢁC, 5% CO2, the opti-
cal densities (OD) were measured at k = 570 nm with a
microtiter plate reader (ANTHOS 2010). The OD values
were used to calculate the T/Ccorr (%) values based on the
following equation T/Ccorr (%) = (ODtest,48 h ꢀ ODblank,t0)/
(ODblank, 48 h ꢀ ODtest, t0) · 100. T/C values between 90%
and 10% were used to construct dose–response curves and
the IC50 values were estimated by linear regression analysis
of T/Cversus the log concentration as previously described.21
3.4.9. Isopropyl 6-hydroxy-2,4-dimethyl-3-(2-(thiophen-
oxy)ethyl)benzoate (6f). The reaction was carried out fol-
lowing the procedure as given for the synthesis of 6a.
Starting with isopropyl 8-hydroxy-4,8-dimethyl-6-
oxospiro[2.5]oct-4-ene-5-carboxylate (4c) (0.035 g, 0.14
mmol), thiophenol (0.020 g, 0.18 mmol), CH2Cl2
(1 mL), and TFA (two drops), 6f was isolated (0.033 g,
70%) as a colorless oil; Rf = 0.64 (hexane/EtOAc = 4:1);
~
IR (neat): m = 2980 (w). 1725 (w), 1654 (s), 1602 (w),
1576 (w), 1467 (m), 1370 (m), 1240 (m), 1100 (m), 740
1
(m) cmꢀ1; H NMR (300 MHz, CDCl3): d = 10.72 (s,
1H, OH), 7.37–7.17 (m, 5 H, ArH), 6.66 (s, 1H, ArH),
5.30 (sep, 1H, J= 6.2 Hz, OCH), 2.96–2.83 (m, 4H,
CH2), 2.42 (s, 3H, CH3), 2.21 (s, 3H, CH3), 1.39 (d,
6H, J= 6.2 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d = 170.9, 159.8, 143.6, 138.4, 136.0 (C), 130.1 (CH),
129.3 (C), 129.0, 128.9, 127.4, 126.4, 117.0 (CH), 112.3
(C), 69.6 (CH), 33.1, 29.6 (CH2), 21.9 (2C), 20.8, 18.5
(CH3); MS (EI; 70 eV) m/z (%) = 344 (M+, 27), 285
(7), 235 (9), 221 (57), 179 (47), 161 (100), 123 (11), 77
(7), 28 (10); elemental analysis: calcd (%) for
C20H24O3S: C, 69.70; H, 7.02; found: C, 69.74; H, 7.20.
Acknowledgments
We thank Sunanda Lahiri for an experimental contribu-
tion. Financial support by the state of Mecklenburg-
Vorpommern (Landesforschungsschwerpunkt ‘Neue
Wirkstoffe und Screeningverfahren’) and by Boehringer
Ingelheim Pharma GmbH & Co. K.G. is gratefully
acknowledged.
3.4.10. Methyl 3-(2-bromoethyl)-2,4-dimethyl-6-vinyl-
benzoate (9). The reaction was carried out following the
procedure as given for the synthesis of 6b. The BF3ÆOEt2
was added last to the reaction mixture. Starting with meth-
yl 8-hydroxy-4,8-dimethyl-6-oxospiro[2.5]oct-4-ene-5-car-
boxylate (4a) (0.030 g, 0.13 mmol), vinylmagnesium
bromide (0.2 mL, 0.20 mmol; 1.0 M solution in THF),
THF (1 mL), and BF3ÆOEt2 (0.01 mL, 0.07 mmol; dis-
solved in 0.2 mL of THF), 7 was isolated (0.013 g, 33%)
as a colorless oil which solidifies upon standing,
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