Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue

Article abstract of DOI:10.1016/S0968-0896(01)00235-8

The syntheses and preliminary evaluation of the first potential bioreductive paclitaxel prodrugs are described. These prodrugs were designed as potential candidates in more selective chemotherapy by targeting hypoxic tumour tissue. Aromatic nitro and azid

Full text of DOI:10.1016/S0968-0896(01)00235-8

Bioorganic & Medicinal Chemistry 10 (2002) 71–77
Synthesis of Novel Paclitaxel Prodrugs Designed for Bioreductive
Activation in Hypoxic Tumour Tissue
Eric W.P. Damen,a Tapio J. Nevalainen,b Toine J.M. van den Bergh,a
Franciscus M.H. de Groota and Hans W. Scheerena,*
aDepartment of Organic Chemistry, NSR Center for Molecular Structure, Design and Synthesis, University of Nijmegen,
Toernooiveld, 6525 ED Nijmegen, The Netherlands
bDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
Received 5 April 2001; accepted 2 July 2001
Abstract—The syntheses and preliminary evaluation of the first potential bioreductive paclitaxel prodrugs are described. These
prodrugs were designed as potential candidates in more selective chemotherapy by targeting hypoxic tumour tissue. Aromatic nitro
and azide groups were used as the bioreductive trigger. Generation of paclitaxel occurs after reduction and subsequent 1,6-elim-
ination or 1,8-elimination. All prodrugs are stable in buffer and indeed give paclitaxel after chemical reduction of the aromatic nitro
or azide functionality. In aerobic cytotoxicity assays several prodrugs exhibit diminished cytotoxicity. These compounds are inter-
esting candidates for further biological evaluation. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
of paclitaxel prodrugs can be achieved by blocking the
C20–OH group, which is important for activity.7 The
paclitaxel prodrugs are designed to release paclitaxel
after reduction of the nitro group to a hydroxylamine or
amine group and subsequent1,6-eliminaiton of a 4-
hydroxylamino or 4-amino benzyloxycarbonyl moiety,
a concepthtathas been used successfully in prodrug
chemistry.8
Many solid tumours are inefficient in the development
of their own blood supply, resulting in hypoxic regions
within the tumour cell population.1 In recentyears, a
number of low toxic (pro)drugs have been developed,
capable of selective activation in hypoxic tissue.2 Many
of these compounds use the reduction of an aromatic
nitro compound as a bioreductive switch to generate the
active species.3 Among these, analogues of nitroimida-
zoles,4 N-oxides5 and nitrobenzyl carbamates6 have
been used as reductive trigger. We now report the first
synthesis of bioreductive prodrugs of paclitaxel 1,
employing the aromatic nitro and azido group as the
bioreductive trigger. Paclitaxel is one of the most pro-
mising anti-cancer agents used in the clinic today.
However, also paclitaxel exhibits dose limiting side
effects, a common problem with cytotoxic agents. Pro-
drug strategies can reduce these side effects by selective
release of the active drug in tumour tissue. Because of
the potency of the parent drug, paclitaxel prodrugs can
be very powerful chemotherapeutic agents. Low toxicity
Synthesis
In a proof of principal study, prodrug 1a was prepared
via reaction of paclitaxel with 4-nitrobenzyl chloro-
formate (PNBC). The compound was stable in buffer
solution (pH=7.4, 37 ꢀC) and upon chemical reduction
of the nitro group formation of paclitaxel was indeed
observed. Next, a series of prodrugs was prepared with
additional electron withdrawing substituents on the
aromatic ring and with nitro heteroaromatic moieties
(which both increase the reduction potential of the nitro
group). Finally some paclitaxel prodrugs were synthe-
sised with an aromatic azide group as the bioreductive
trigger. Azides are known to undergo oxygen-inhibited
enzymatic reduction9 and can therefore potentially serve
to trigger hypoxia selective prodrugs.10
*Corresponding author. Tel.: +31-24-365-2331; fax: +31-24-365-
2929; e-mail: jsch@sci.kun.nl
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00235-8

72
E. W. P. Damen et al. / Bioorg. Med. Chem. 10 (2002) 71–77
The prodrugs 1b–1i were prepared in good yield by
activation of the alcohols (3b–3i)11 with 4-nitrophenyl
chloroformate and subsequent coupling of the activated
carbonates (2b–i) with paclitaxel (Scheme 1).
After reduction (NaBH4, EtOH) and elimination, a pri-
0
zoyl14 atC2 to the C30–NH2 resulted in the formation
of paclitaxel.
mary amine0 was formed atC3 . Migration of the ben-
Prodrug 1c contains of a novel self-eliminating moiety,
that after chemical reduction of the nitro group (Zn,
AcOH/MeOH) fragmented to give the parent drug in
quantitative yield via 1,8-elimination (Scheme 2). The
other prodrugs also yielded paclitaxel after chemical
reduction.
Biological Activity
All prodrugs proved to be stable in Tris–buffer
(pH=7.4, 37 ꢀC) for at least 24 h. Cytotoxicity assays
againstseven well defined human utmour cell lines
(Table 1) showed that prodrugs 1a–b and 1d–i possess
only a slightly reduced cytotoxicity. This is probably
due to enzymatic hydrolysis of the benzyl carbonate by
esterases present in the assay. This results in nonspecific
release of paclitaxel. Lack of stability is a major
problem for many paclitaxel prodrugs.15
A different strategy was applied in the design and
synthesis of azide prodrug 1k in which the 30NH-posi-
tion of paclitaxel is functionalised with the self elim-
inating moiety and the C20–OH position is blocked by a
benzoyl group. This prodrug was prepared through
semisynthesis by treating optically active phenylisoser-
ine methyl ester with 4-azidobenzyl-40-nitrophenyl car-
bonate. The modified side chain was protected as an
oxazolidine12 prior to coupling. Saponification of the
methyl ester and coupling to 7-Aloc baccatin III13 yiel-
ded the protected prodrug. After deprotection and
benzoylation prodrug 1k was obtained (Scheme 3).
Prodrug 1c displayed a significantly reduced cytotoxi-
city and therefore a greater stability towards hydrolytic
enzymes. The novel linker of prodrug 1c mightbe very
useful as a spacer in the preparation of paclitaxel pro-
drugs with targets other than hypoxia, since prodrug
lability is often a problem with tumour selective
Scheme 1.
Scheme 2.

E. W. P. Damen et al. / Bioorg. Med. Chem. 10 (2002) 71–77
73
Scheme 3. (i) 4-Azidobenyl-40-nitrophenyl carbonate, pyridine, THF, 3 hm rtm 70%; (ii) 4-methoxybenzaldehyde dimethyl acetal, PTS, toluene,
reflux, 3 h, 97%; (iii) K2CO3, MeOH, H2O, 16 h, rt, 76%; (iv) 7-Aloc baccatin III DIC, DMAP, CH2Cl2, rt, 3 h 59%; (v) Pd(PPh3)4, morpholine,
THF, rt15 min, 78%; (vi) PTS, MeOH, rt24 h, 87%; (vii) benzoic acid, DIC, DMAP, CH 2Cl2, rt, 4 h, 76%.
Table 1. Cytotoxicity assay of paclitaxel prodrugs against seven well
defined human tumour cell lines
Elemental analyses were carried out in triplicate on a
Carlo Erba EA 1108 element analyzer. Melting points
were measured on a ReichertThermopan microscope
and are uncorrected. TLC analysis was performed on
Merck precoated silica gel 60 F-254 plates. Spots were
visualized with UV light and 10% H2SO4 (1 L) solution
containing ammonium molybdate (25 g) and ceric
ammonium sulfate (10 g) followed by charring. Column
chromatography was preformed with silica 60 (Baker).
THF was dried by refluxing over sodium and was dis-
tilled prior to use. Dichloromethane was distilled from
calcium hydride prior to use. When necessary, all reac-
tions were carried out under an argon atmosphere.
Unless stated otherwise, materials were obtained from
commercial sources and used without further purifica-
tion. Paclitaxel was generous gift of Pharmachemie,
Haarlem, The Netherlands.
a
Compound
IC50 (ng/ mL)
H226 MCF7 EVSA-T WIDR IGROV M19 A498
Paclitaxel
<3
8
6
187
7
7
19
13
5
6
14
67
<3
4
<3
186
10
<3
6
5
<3
4
3
10
36
36
620
83
114
19
13
5
<3
9
7
384 1387
101
151
25
14
7
<3
52
50
1a
1b
1c
1d
1e
1f
1g
1h
1i
221
19
21
5
<3
<3
<3
6
308
19
18
6
<3
<3
<3
4
83
141
66
36
22
21
41
193
11
5
<3
<3
<3
<3
25
6
23
158
9
14
68
1j
1k
53
41
aIC50, drug concentration required to inhibit cell proliferation to 50%
versus untreated cells (37 ꢀC, 5 days).
20-(4-Nitrobenzyl carbonate) paclitaxel (1a). To a solu-
tion of paclitaxel (500 mg, 0.60 mmol) in CH2Cl2 (75
mL) was added diisopropyl ethylamine (220 mL, 1.21
mmol), 4-nitrobenzyl chloroformate (156 mg, 0.72
mmol) and a few crystals of DMAP. After stirring for 3
h, with not all the starting material consumed, the mix-
ture was diluted with EtOAc and washed with a satu-
rated NaHCO3 solution, demineralised water, 0.5 N
KHSO4 solution and brine. The solution was dried over
Na2SO4 and concentrated in vacuo. The residue was
purified via column chromatography (EtOAc/hexane
1:1), affording the title compound 1a (405 mg, 0.39
mmol, 67% (conversion yield 89%) and unreacted
paclitaxel (138 mg, 0.16 mmol). Mp 170–171 ꢀC; 1H
NMR (300 MHz, CDCl3) d 8.22 (d, 2H, J=8.7 Hz,
nitrophenyl), 8.15 (d, 2H, J=7.2 Hz, H–Ph), 7.72 (d,
2H, J=7.5 Hz, H–Ph), 7.46 (m, 13H, H–Ph, nitrophe-
nyl), 6.86 (d, 1H, J=9.4 Hz, NH), 6.29 (m, 2H, H10,
H13), 6.02 (dd, 1H, J=9.3, 2.7 Hz, H30), 5.69 (d, 1H,
J=7.1 Hz, H2), 5.46 (d, 1H, J=2.8 Hz, H20), 5.30 (d,
1H, J=13.2 Hz, Ar–CH2), 5.23 (d, 1H, J=13.2 Hz, Ar–
CH2), 4.98 (d, 1H, J=7.7 Hz, H5), 4.44 (m, 1H, H7),
4.33 (d, 1H, J=8.5 Hz, H20a), 4.21 (d, 1H, J=8.4 Hz,
H20b), 3.82 (d, 1H, J=7.0 Hz, H3), 2.53 (m, 1H, H6a),
2.47 (s, 3H, 4-OAc), 2.35 (m, 2H, H14), 2.24 (s, 3H, 10-
OAc), 1.92 (s, 3H, H18), 1.90 (m, 1H, H6b), 1.69 (s, 3H,
H19), 1.26 (s, 3H, H16), 1.14 (s, 3H, H17); FAB-MS,
1033 [M+H]+, 1055 [M+Na]+. Anal. (C55H56N2O18)
paclitaxel prodrugs. Prodrug 1k showed a lower toxicity
as well, although less apparently than prodrug 1c.
Compound 1j, which can be considered a new paclitaxel
derivative modified at C30-NH, showed a slightly lower
cytotoxicity compared to paclitaxel.
Conclusions
In conclusion, we have prepared the first bioreductive
paclitaxel prodrugs. All prodrugs were stable in buffer
and released paclitaxel after reduction. Unfortunately,
benzyl carbonate prodrugs 1a–b and 1d–i did notshow
a much reduced cytotoxicity under aerobic conditions.
Prodrugs 1c and 1k did display a lower cytotoxicity and
these are selected for further evaluation in anaerobic
environment.
Experimental
1H NMR spectra were recorded on a Bruker AM-300
(300 MHz) or on a Bruker AC100 (100 MHz) spectro-
meter. Chemical shift values are given in ppm (d) rela-
tive to TMS as internal standard. For numbering of the
atoms in paclitaxel, see Scheme 1. Mass spectra were
obtained with VG Micromass 7070E spectrometer.

74
E. W. P. Damen et al. / Bioorg. Med. Chem. 10 (2002) 71–77
1
calcd C 63.95%, H 5.46%, N 2.71%, measured C
64.11%, H 5.42%, N 2.64%.
dine 6 (642 mg, 1.32 mmol, 97%); mp 123–126 ꢀC; H
NMR (100 MHz, CDCl3) d 7.34 (m, 7H), 6.80 (m, 6H),
6.40 (s, 1H), 5.48 (d, 1H, J=4.1 Hz), 4.93 (d, 1H,
J=12.2 Hz), 4.66 (d, 1H, J=12.2 Hz), 4.58 (d, 1H,
J=4.1 Hz), 3.81 (s, 3H), 3.58 (s, 3H).
20-(4-Azidobenzyl carbonate) paclitaxel (1b). A solution
of 4-azidobenzyl alcohol (22.9 mg, 0.154 mmol), pyri-
dine (16.0 mL, 0.200 mmol), 4-nitrophenyl chloro-
formate (27.8 mg, 0.138 mmol) and a few crystals of
DMAP in CH2Cl2 (5 mL) was leftsitrring for 40 min.
After no 4-azidobenzyl alcohol could be detected by
means of TLC (SiO2, CH2Cl2/MeOH 95:5), paclitaxel
(100.4 mg, 0.118 mmol) and DMAP (16.2 mg, 0.133
mmol) were added and stirring was continued for 70 h.
The mixture was diluted with EtOAc and subsequently
washed with a saturated NaHCO3 solution, deminer-
alised water, aqueous 0.5 N KHSO4 solution and brine.
The solution was dried over Na2SO4 and concentrated
in vacuo. The residue was purified by column chroma-
tography (EtOAc/hexane 1:1), to yield prodrug 1b
(121.0 mg, 0.118 mmol, 100%); mp 139–142 ꢀC; 1H
NMR (300 MHz, CDCl3) d 8.15 (d, 2H, J=7.1 Hz, H–
Ph), 7.72 (d, 2H, J=7.1 Hz, H–Ph), 7.61 (t, 1H, J=7.3
Hz, H–Ph), 7.47 (m, 12H, H–Ph, azidophenyl), 7.01 (d,
2H, J=8.4 Hz, azidophenyl), 6.88 (d, 1H, J=9.3 Hz,
NH), 6.30 (m, 2H, H10, H13), 5.98 (dd, 1H, J=9.2 Hz,
2.7 Hz, H30), 5.69 (d, 1H, J=7.1 Hz, H2), 5.44 (d, 1H,
J=2.7 Hz, H20), 5.30 (d, 1H, J=13.2 Hz, Ar–CH2a), 5.23
(d, 1H, J=13.2 Hz, Ar–CH2b), 4.98 (d, 1H, J=7.7 Hz,
H5), 4.45 (m, 1H, H7), 4.33 (d, 1H, J=8.5 Hz, H20a), 4.21
(d, 1H, J=8.4 Hz, H20b), 3.82 (d, 1H, J=7.1 Hz, H3),
2.57 (m, 1H, H6a), 2.46 (s, 3H, 4-OAc), 2.35 (m, 2H, H14),
2.24 (s, 3H, 10-OAc), 1.92 (s, 3H, H18), 1.89 (m, 1H, H6b),
1.69 (s, 3H, H19), 1.24 (s, 3H, H16), 1.14 (s, 3H, H17);
FAB-MS, 1029 [M+H]+, 1051 [M+Na]+. Anal.
(C55H56N4O16) calcd C 64.19%, H 5.49%, N 5.44%,
measured C 64.25%, H 5.56%, N 5.26%.
N-(4-Azidobenzyloxycarbonyl) oxazolidine carboxylic
acid (7). The oxazolidine methyl ester 6 (639 mg, 1.31
mmol) was dissolved in MeOH (15 mL). Next, a solu-
tion of K2CO3 (452 mg, 3.28 mmol) in demineralised
water (5 mL) was added. The reaction was stirred for 16
h at room temperature. The reaction mixture was dilu-
ted with water and washed with Et2O to remove organic
impurities. Acetate buffer (pH=5) was added to the
water layer, which was extracted 3 times with CH2Cl2.
The solution was dried over Na2SO4 and concentrated
in vacuo to yield the title compound, which is used in
the following reaction without further purification. The
yield of compound 7 was 472 mg (1.00 mmol, 76%); mp
162–166 ꢀC; 1H NMR (300 MHz, CDCl3) d 7.34 (m,
7H), 6.87 (m, 2H), 6.79 (d, 2H, J=8.5 Hz), 6.71 (d, 2H,
J=8.5 Hz), 6.41 (s, 1H), 5.47 (d, 1H, J=4.1 Hz), 4.91
(d, 1H, J=12.2 Hz), 4.68 (d, 1H, J=12.3 Hz), 4.63 (d,
1H, J=4.2 Hz), 3.81 (s, 3H).
7-Aloc-20,30-oxazolidine protected derivative 8. To a
solution of 7-Aloc baccatin III (195 mg, 0.29 mmol) in
CH2Cl2 (10 mL) at0 ꢀC was subsequently added: oxa-
zolidine carboxylic acid 7 (276 mg, 0.58 mmol), DIC
(112 mL, 0.73 mmol) and a few crystals of DMAP. The
reaction was stirred at room temperature. After all the
7-Aloc baccatin III was consumed, as indicated on
TLC, the mixture was diluted with EtOAc and washed
with a saturated NaHCO3 solution, demineralised
water, 0.5 N KHSO4 solution and brine. The solution
was dried over Na2SO4 and evaporated to dryness.
Column chromatography (EtOAc/heptane 1:1) yielded
compound 8 (194 mg, 0.17 mmol, 59%); mp 104–
106 ꢀC; This compound was used without further pur-
ification in the next step. 1H NMR (300 MHz, CDCl3) d
8.02 (d, 2H, J=7.4 Hz, H–Ph), 7.63 (t, 1H, J=7.4 Hz,
H–Ph), 7.40–7.12 (m, 9H, H–Ar), 6.89 (d, 2H, J=6.7
Hz, azidophenyl), 6.78 (d, 2H, J=8.4 Hz, H–Ph), 6.69
(d, 2H, J=8.0 Hz, H–Ph–OMe), 6.41 (s, 1H, oxazoli-
dine), 6.19 (s, 1H, H10), 6.10 (t, 1H, J=8.3 Hz, H13),
5.95 (m, 1H, H30), 5.61 (d, 1H, J=7.0 Hz, H2), 5.42 (m,
H, H20, allyl), 5.30 (m, 2H, Ar–CH2), 4.88 (m, 2H,
allyl), 4.63 (m, 3H, allyl, H7), 4.24 (d, 1H, J=8.4 Hz,
H20a), 4.08 (d, 1H, J=8.3 Hz, H20b), 3.84 (d, 1H,
J=4.4 Hz, H3), 3.83 (s, 3H, MeO), 2.56 (m, 1H, H6a),
2.46 (s, 3H, 4-OAc), 2.33 (m, 2H, H14), 2.13 (s, 3H, 10-
OAc), 1.90 (m, 1H, H6b), 1.82 (s, 3H, H18), 1.62 (s, 3H,
H19), 1.20 (s, 3H, H16), 1.13 (s, 3H, H17).
(2R,3S)-N-(4-Azidobenzyloxycarbonyl) phenylisoserine
methyl ester (5). A solution of amino alcohol 416 (0.50
g, 2.6 mmol) in THF (15 mL), pyridine (337 mL, 4.2
mmol) and 4-azidobenzyl,40-nitrophenyl carbonate
(0.82 g, 2.6 mmol) was stirred for 72 h in the absence of
light. The mixture was diluted with EtOAc and washed
with a saturated NaHCO3 solution, demineralised
water, 0.5 N KHSO4 solution and brine. The solution
was dried over Na2SO4 and concentrated in vacuo.
Crystallisation in a hexane/EtOAc mixture yielded
compound 5 (0.67 g, 1.8 mmol, 69%); mp 67–70 ꢀC; H
1
NMR (100 MHz, CDCl3) d 7.35 (m, 7H), 6.96 (m, 3H),
5.69 (d, 1H, J=9.0 Hz), 5.25 (d, 1H, J=9.0 Hz), 5.04 (s,
2H), 4.48 (br s, 1H), 3.81 (s, 3H).
N-(4-Azidobenzyloxycarbonyl) oxazolidine methyl ester
(6). To a solution of 5 (502 mg, 1.36 mmol) in anhy-
drous toluene (20 mL) was added 4-methoxy-
benzaldehyde dimethyl acetal (1.25 mL, 6.80 mmol) and
a few crystals of PTS. The reaction was heated to reflux
and stirring was continued for 30 min. The reaction was
allowed to cool to ambient temperature and the reaction
mixture was quenched with NaHCO3 and diluted with
Et2O. The organic layer was washed with washed with a
saturated NaHCO3 solution, demineralised water and
brine. The resulting yellow oil was purified via column
chromatography (EtOAc/heptane 2:5) to yield oxazoli-
30N-(4-Azidobenzyloxycarbonyl)-30N-debenzoyl
pacli-
taxel (1j). To a solution of 8 (147 mg, 0.13 mmol) and
morpholine (64 mL, 0.73 mmol) in THF (5 mL), a few
crystals of Pd(PPh3)4 were added. When the reaction
was complete, as was demonstrated by TLC (EtOAc/
hexane 1:1), the mixture was filtered over Hyflo. The
filtrate was concentrated in vacuo. The residue was
taken up in EtOAc and successively washed with a
saturated NaHCO3 solution, demineralised water, 0.5 N

E. W. P. Damen et al. / Bioorg. Med. Chem. 10 (2002) 71–77
75
KHSO4 solution and brine. The organic layer was dried
over Na2SO4, filtered and concentrated in vacuo. The
residue was purified via column chromatography
(EtOAc/hexane 1:1). The total yield of this reaction was
dissolved in MeOH (15 mL). To this solution PTS (19.8
mg, 0.104 mmol) was added. The mixture was stirred in
the absence of light in for 24 h, concentrated, diluted
with EtOAc and washed with a NaHCO3 solution and
brine. The solution was dried over Na2SO4 and evapo-
rated to dryness. Column chromatography (CH2Cl2/
MeOH 95:5) and freeze drying in dioxane afforded 1j
(70.1 mgꢀ, 0.075 mmol, 87%) as a fluffy white solid; mp
146–151 C; 1H NMR (300 MHz, CDCl3) d 8.12 (d, 2H,
J=7.6 Hz, H–Ph), 7.51 (t, 1H, J=7.6 Hz, H–Ph), 7.41
(m, 8H, H–Ar), 7.17 (d, 2H, J=8.3 Hz, H–Ar), 6.92 (d,
2H, J=6.7 Hz, azidophenyl), 6.27 (s, 1H, H10), 6.23 (t,
1H, J=8.5 Hz, H13), 5.65 (m, 2H, H30, H2), 5.03 (d,
1H, J=12.2 Hz, Ar–CH2a), 4.94 (d, 1H, J=7.2 Hz,
H5), 4.91 (d, 1H, J=12.1 Hz, Ar–CH2b), 4.66 (br s, 1H,
H20), 4.40 (m, 1H, H7), 4.30 (d, 1H, J=8.4 Hz, H20a),
4.18 (d, 1H, J=8.3 Hz, H20b), 3.79 (d, 1H, J=7.1 Hz,
H3), 2.56 (m, 1H, H6a), 2.45 (s, 3H, 4-OAc), 2.17 (s,
3H, 10-OAc), 2.12 (m, 2H, H14), 1.92 (m, 1H, H6b),
1.88 (s, 3H, H18), 1.65 (s, 3H, H19), 1.24 (s, 3H, H16),
1.15 (s, 3H, H17); FAB-MS, 947 [M+Na]+. Anal.
(C48H52N4O15) calcd C 62.33%, H 5.67%, N 6.06%,
measured C 62.34%, H 5.82%, N 5.36%.
5.46 (d, 1H, J=2.8 Hz, H20), 4.99 (bd, 1H, J=7.9 Hz,
H5), 4.87 (bt, 2H, CH2-spacer), 4.39 (m, 1H, H7), 4.32
(d, 1H, J=8.4 Hz, H20a), 4.26 (d, 1H, J=8.4 Hz,
H20b), 3.82 (d, 1H, J=7.0 Hz, H3), 2.55 (m, 1H, H6a),
2.46 (s, 3H, 4-OAc), 2.22 (s, 3H, 10-OAc), 1.96 (s, 3H,
H18), 1.70 (s, 3H, H19), 1.22 (s, 3H, H16), 1.17 (s, 3H,
H17); FAB-MS, 1059 [M+H]+, 1081 [M+Na]+. Anal.
1
.
(C57H58N2O18 2 /2H2O) calcd C 62.01%, H 5.75%, N
2.54%, measured C 62.06%, H 5.31%, N 2.60%.
Proof of principle of 1,8-elimination: chemical reduction
of the nitrocinnamyl carbonate 1c. 36 mg of the prodrug
20-[4-nitrocinnamyl carbonate]-paclitaxel 1c was dis-
solved in 8 mL methanol and 2 mL acetic acid. A cata-
lytic amount of Zinc powder was added and the red
mixture was stirred for 12 h. Dichloromethane was
added and the organic layer was washed with saturated
sodium bicarbonate, 0.5 N potassium bisulfate, brine,
and water and dried over anhydrous sodium sulfate.
After evaporation of the solvents the residual yellow
film was purified by means of column chromatography
(ethyl acetate/hexane; 2:1), to yield 28 mg of paclitaxel
1
(confirmed by 300 MHz H NMR) and 4 mg of unreac-
ted starting compound. When the compound was stir-
red in the absence of zinc powder under the same
conditions, no paclitaxel was formed, indicating that
reduction of the nitro group by zinc induced the release
of paclitaxel.
Azido prodrug 1k. To a solution of 1j (55.0 mg, 0.060
mmol) in CH2Cl2 (10 mL) were added DIC (20 mL, 0.13
mmol) and a few crystals of DMAP. After stirring for 4
h, the mixture was diluted with EtOAc and washed with
a saturated NaHCO3 solution, demineralised water,
0.5 N KHSO4 solution, again with demineralised water
and brine. The solution was dried over Na2SO4 and
evaporated to dryness. The residue was purified via col-
umn chromatography (EtOAc/hexane 2:3), affording
prodrug 1k (47.8 mg, 0.046 mmol, 77%); mp 150–
20-O-(2,4-Dinitrobenzyloxycarbonyl) paclitaxel (1d). The
procedure identical to the preparation of prodrug 1b
from 3d17,18 was followed to yield 1d (101 mg, 0.09
mmol, 78%) as a white solid; mp 161–163 ꢀC; H NMR
1
(300 MHz, CDCl3) d 9.00 (1H, d, J=1.8 Hz, H3–Ph),
8.47 (1H, dd, J=2.6, J=8.8 Hz, H5–Ph), 8.16 (2H, d,
J=7.3 Hz, H–Ph), 7.83 (1H, d, J=8.6 Hz, H–Ph), 7.74
(2H, d, J=7.5 Hz, H–Ph), 7.61 (1H, m, H–Ph), 7.54–
7.36 (10H, m, H–Ph), 6.89 (1H, d, J=9.7 Hz, NH), 6.28
(1H, s, H10), 6.28 (1H, m, H13), 6.01 (1H, dd, J=2.6,
9.7 Hz, H30), 5.70 (1H, d, J=7.2 Hz, H2), 5.44 (1H, d,
J=2.6 Hz, H20), 5.17 (2H, s, Ar–CH2), 4.98 (1H, dd,
J=9.7, 2.6 Hz, H5), 4.44 (1H, dd, J=11.4, 7.0 Hz, H7),
4.32 (1H, d, J=8.8 Hz, H20a), 4.21 (1H, d, J=8.8 Hz,
H20b), 3.82 (1H, d, J=6.1 Hz, H3), 2.56 (1H, ddd,
J=6.1, 9.7, 14.9 Hz, H6a), 2.47 (3H, s, 4-OAc), 2.43
(1H, m, H14a), 2.40 (1H, m, C7OH), 2.23 (2H, m,
H14b, H6b), 2.22 (3H, s, 10-OAc), 1.93 (3H, s, H18),
1.87 (1H, m, H6b), 1.69 (3H, s, H19), 1.25 (3H, s, H16),
1.15 (3H, s, H17); FAB-MS, 1100 [M+Na]+. Anal.
(C55H55N3O20) calcd C 61.3%, H 5.1%, N 3.9%, mea-
sured C 61.1%, H 4.6%, N 3.7%.
ꢀ
1
152 C; H NMR (300 MHz, CDCl3) d=8.11 (d, 2H,
J=7.4 Hz, H–Ph), 7.96 (d, 2H, J=8.2 Hz, azidophe-
nyl), 7.51–7.21 (m, 11H, H–Ar), 7.17 (d, 2H, J=7.9 Hz,
H–Ph), 6.89 (d, 1H, J=8.3 Hz, NH), 6.28 (m, 2H, H10,
H13), 5.65 (m, 4H, H2, H20, Ar–CH2), 5.05 (d,
1H,J=12.4 Hz, Ar–CH2a), 4.96 (d, 1H, J=6.5 Hz, H5),
4.93 (d, 1H, J=12.2 Hz, ArCH2b), 4.48 (m, 1H, H7),
4.29 (d, 1H, J=8.4 Hz, H20a), 4.18 (d, 1H, J=8.4 Hz,
H20b), 3.79 (d, 1H, J=7.1 Hz, H3), 2.56 (m, 1H, H6a),
2.46 (s, 3H, 4-OAc), 2.33 (m, 2H, H14), 2.23 (s, 3H, 10-
OAc), 1.88 (s, 3H, H18), 1.90 (m, 1H, H6b), 1.67 (s, 3H,
H19), 1.23 (s, 3H, H16), 1.13 (s, 3H, H17); FAB-MS,
1029
[M+H]+,
1051
[M+Na]+.
Anal.
.
(C55H56N4O16 H2O) calcd C 63.10%, H 5.55%, N
5.34%, measured C 63.56%, H 5.59%, N 5.32%.
3-Bromomethyl-4-nitrobenzoic acid ethyl ester. The mix-
ture of 3-methyl-4-nitrobenzoic acid ethyl ester19 (5.0 g,
24 mmol), NBS (6.4 g, 36 mmol), (BzO)2, in CCl4 (100
mL) was refluxed and irradiated with 250 W lamp for 16
h. The solvent was evaporated and the residue was
chromatographed on silica gel eluting with EtOAc/hep-
tane 1:8 to afford the title compound as an oil (4.87 g,
17 mmol, 70%), which was used as such in the next step;
1H NMR (100 MHz, CDCl3) d 8.22 (1H, m, H3), 8.10
(2H, m, H4, H6), 4.83 (2H, s), 4.44 (2H, q, J=7.1 Hz),
1.43 (3H, t, J=7.1 Hz).
20-(4-Nitrocinnamylcarbonate) paclitaxel (1c). The pro-
cedure identical to the preparation of prodrug 1b was
followed to yield 1c (58%); mp 151 ꢀC; 1H NMR
(300 MHz, CDCl3) d=8.19 (d, 2H, J=8.7 Hz, nitro-
phenyl), 8.15 (d, 2H, J=7.2 Hz, H–Ph), 7.75 (d, 2H,
J=7.2 Hz, H–Ph), 7.35–7.67 (m, 13H, H–Ph), 6.75 (d,
1H, J=16.0 Hz, HC=CH–CH2), 6.43 (dt, 1H, J=16.0
Hz, HC=CH–CH2), 6.34 (s, 1H, H10), 6.26 (bt, 1H,
H13), 6.01 (m, 1H, H30), 5.72 (d, 1H, J=7.1 Hz, H2),

76
E. W. P. Damen et al. / Bioorg. Med. Chem. 10 (2002) 71–77
3-Hydroxymethyl-4-nitrobenzoic acid ethyl ester. The
mixture of 3-bromomethyl-4-nitrobenzoic acid ethyl
ester (1.6 g, 5.5 mmol), H2SO4 (3 mL) and water (50
mL) was refluxed overnight. The solution was made
basic with 5 N NaOH solution and extracted with die-
thyl ether. The aqueous layer was acidified with 1 N
HCl, extracted into EtOAc, dried (Na2SO4) and evapo-
rated. The residue was refluxed in EtOH (150 mL) with
H2SO4 (1 mL) overnight. EtOH was evaporated and the
residue was chromatographed on silica gel eluting with
EtOAc/heptane 2:5 to afford the title compound as an
oil (1.1 g, 89%), which was directly used in the follow-
H20b), 3.81 (1H, d, J=7.0 Hz, H3), 2.55 (1H, m, H14a),
2.46 (3H, s, 4-OAc), 2.52–2.36 (3H, m, C7OH, H6a and
H14b), 2.23 (3H, s, 10-OAc), 1.91 (3H, s, H18), 1.89
(1H, m, H6b), 1.68 (3H, s, H19), 1.24 (3H, s, H16), 1.14
(3H, s, H17); FAB-MS, 1139 [M+Na]+. Anal.
(C59H60N2O20) calcd C 63.4%, H 5.4%, N 2.5%,
measured C 63.2%, H 5.1%, N 2.7%.
20-O-(2-Nitro-5-carboxybenzyloxycarbonyl)paclitaxel (1f).
To a solution of (78 mg, 0.071 mmol) in dry THF
under an argon was added glacial acetic acid (10 mL,
0.18 mmol, 2.5 equiv) and (C4H9)3SnH and few crystals
of Pd(PPh3)4. After 30 min 1 mL of 0.5 M HCl in
EtOAc was added carefully with stirring. 10 mL ethyl
ether and 20 mL EtOAc were added and the organic
phase was washed with water, brine, dried (Na2SO4)
and evaporated. The crude compound was chromato-
graphed with silica gel eluting first with CH2Cl2 and then
MeOH/CH2Cl2 (1:9) to afford 1f as white solid (49.2 mg,
0.046 mmol, 64%). 1H NMR (300 MHz, CDCl3/
CD3OD) d 8.31 (1H, s, H–Ph), 8.17 (2H, s, H–Ph), 8.14
(2H, d, J=7.8 Hz, H–Ph), 7.75 (2H, d, J=6.8 Hz, H–
Ph), 7.60 (1H, m, H–Ph), 7.52–7.36 (10H, m, H–Ph), 6.31
(1H, s, H10), 6.22 (1H, bt, J=8.7 Hz, H13), 6.01 (1H, dd,
J=3.0, 9.2 Hz, H30), 5.70 (1H, d, J=7.1 Hz, H2), 5.65
(2H, d, J=15.1 Hz, Ar–CH2a), 5.57 (2H, d, J=15.1 Hz,
Ar–CH2b), 5.50 (1H, d, J=3.5 Hz, H20), 4.97 (1H, bd,
J=7.1 Hz, H5), 4.39 (1H, dd, J=6.2 Hz, 10.5 Hz, H7),
4.32 (1H, d, J=8.5 Hz, H20a), 4.23 (1H, d, J=8.5 Hz,
H20b), 3.79 (1H, d, J=7.1 Hz, H3), 2.53 (1H, m, H6a ),
2.41 (3H, s, 4-OAc), 2.41–2.34 (2H, m, H14a and C7OH),
2.27 (3H, m, H6 and H14b), 2.23 (3H, s, 10-OAc), 1.89
(3H, s, H18), 1.89 (1H, m, H6b), 1.68 (3H, s, H19), 1.22
(3H, s, H16), 1.15 (3H, s, H17); FAB-MS, 1100
1
ing step; H NMR (100 MHz, CDCl3) d 8.43 (1H, s,
H3), 8.12 (2H, s, H4, H6), 5.03 (2H, s), 4.44 (2H, q,
J=7.2 Hz ), 2.00 (1H, bs, OH), 1.43 (3H, t, J=7.2 Hz).
3-Hydroxymethyl-4-nitrobenzoic acid allyl ester (3e).
The mixture of 3-hydroxymethyl-4-nitrobenzoic acid
ethyl ester (1.0 g, 4.44 mmol), allyl alcohol (100 mL)
and concd H2SO4 (3 mL) was refluxed for 24 h. Allyl
alcohol was evaporated and the residue was purified by
silica gel column chromatography eluting with EtOAc/
heptane 2:5 to afford yellow solid, which was crystal-
lised from isopropanol/heptane to yield 3e as yellow
crystals (270 mg, 1.14 mmol, 26%), which were used as
such in the next step; mp 73–75 ꢀC; 1H NMR (100 MHz,
CDCl3) d 8.46 (1H, s, H3), 8.14 (2H, s, H4, H6), 5.52–
5.29 (2H, m, allyl), 6.20–5.87 (1H, m, allyl), 5.02 (2H,
bs), 5.85 (2H, m, allyl), 2.51 (1H, bs, OH).
2-Nitro-5-(allyloxycarbonyl)benzyl 4-nitrophenyl carbon-
ate (2e). To a mixture of 3e (170 mg, 0.72 mmol), pyri-
dine (0.28 mL) in dry THF (10 mL) was added dropwise
4-nitrophenyl chloroformate (217 mg, 1.07 mmol) in dry
THF (10 mL) at20 ꢀC and the mixture was stirred for
42 h. The reaction mixture was filtered and evaporated
under reduced pressure. The oily residue was redis-
solved in EtOAc (20 mL), washed with 10% citric acid
solution and brine, dried (Na2SO4) and evaporated to
give oil, which was purified by silica gel column chro-
matography eluting with CH2Cl2/heptane 3:1 to afford
2e as white solid (171mg, 0.42 mmol, 59%), which was
used directly in the subsequent reaction step; mp 102–
104 ꢀC; 1H NMR (100 MHz, CDCl3) d 8.39 (1H, s, H3),
8.31 (2H, m, H30,50), 8.31 (2H, s), 8.23 (2H, s, H4, H6),
7.42 (2H, m, H20,60), 4.90 (2H, d, J=5.7 Hz), 5.52–5.29
(2H, m, allyl), 5.74 (2H, s, CH2O), 6.20–5.87 (1H, m,
allyl), 5.02 (2H, bs), 5.85 (2H, m, allyl) 2.51 (1H, bs,
OH).
[M+Na+1]+. Anal. (C55H56N2O20 2H2O) C 60.4%, H
.
5.4%, N 2.5%, measured C 60.2%, H 5.4%, N 2.4%.
20-O-(5-Methyl-nitro-1H-imidazol-2-yl)methyloxycarbo-
nyl)paclitaxel (1g). Analogously to the preparation of
1b from 3g20 to afford 1g as a white powder (101 mg,
91%); mp 150–152 ꢀC; 1H NMR (300 MHz, CDCl3)
d=8.15 (2H, d, J=7.2 Hz, H–Ph), 7.72 (2H, d, J=7.3
Hz, H–Ph), 7.61 (1H, m, H–Ar), 7.54–7.35 (10H, m, H–
Ph), 7.26 (1H, d, J=3.5 Hz, H–Ar), 6.88 (1H, d, J=8.8
Hz, NH), 6.66 (1H, d, J=3.5 Hz, H–Ar), 6.30 (1H, s,
H10), 6.28 (1H, d, J=9.7 Hz, H13), 6.01 (1H, dd,
J=2.6 Hz, 9.7 Hz, H30), 5.70 (1H, d, J=7.2 Hz, H2),
5.44 (1H, d, J=2.6 Hz, H20), 5.17 (2H, s, Ar–CH2), 4.98
(1H, dd, J=9.7 Hz, 2.6 Hz, H5), 4.44 (1H, dd, J=11.4
Hz, 7.0 Hz, H7), 4.32 (1H, d, J=8.8 Hz, H20a), 4.21
(1H, d, J=8.8 Hz, H20b), 3.82 (1H, d, J=6.1 Hz, H3),
2.56 (1H, ddd, J=6.1 Hz, 9.7 Hz, 14.9 Hz, H6a ), 2.47
(3H, s, 4-OAc), 2.47–2.35 (2H, m, C7OH, H14b), 2.25
(1H, m, H14a) 2.24 (3H, s, 10-OAc), 1.93 (3H, s, H18),
1.83 (1H, m, H6b), 1.69 (3H, s, H19), 1.25 (3H, s, H16),
1.15 (3H, s, H17); FAB-MS, 1037[M+H]+, 1059
20-O-[2-Nitro-5-(allyloxycarbonyl)benzyloxycarbonyl]pa-
clitaxel (1e). Analogously to the preparation of 1b to
afford 1e as white solid (159 mg, 90%); 1H NMR
(300 MHz, CDCl3) d 8.32 (1H, s, H–Ph), 8.17 (2H, s, H–
Ph), 8.15 (2H, d, J=7.3 Hz, H–Ph), 7.76 (2H, d, J=7.4
Hz, H–Ph), 7.60 (1H, m, H–Ph), 7.54–7.34 (10H, m, H–
Ph), 6.94 (1H, d, J=9.7 Hz, NH), 6.28 (1H, s, H10),
6.28 (1H, m, H13), 6.03 (1H, dd, J=2.6, 8.7 Hz, H30),
5.97 (1H, m, allyl), 5.69 (1H, d, J=7.2 Hz, H2), 5.60
(2H, s, Ar–CH2), 5.48 (1H, d, J=2.6 Hz, H20), 5.43–
5.27 (2H, m, allyl), 4.98 (1H, dd, J=9.7, 2.6 Hz, H5),
4.85 (2H, d, J=6.5 Hz, allyl), 4.43 (1H, m, H7), 4.32
(1H, d, J=8.3 Hz, H20a), 4.20 (1H, d, J=8.3 Hz,
[M+Na]+. Anal. (C53H56N4O18 H2O) calcd C 60.34%,
.
H 5.54%, N 5.31%, measured C 60.23%, H 5.68%, N
4.95%.
20 - O - (5 - Nitrofuran - 2 - ylmethyloxycarbonyl)paclitaxel
(1h). Analogously to the preparation of 1b from 3h21 to
yield 1h as a white powder (90 mg, 74%); mp 156–158 ꢀC.

E. W. P. Damen et al. / Bioorg. Med. Chem. 10 (2002) 71–77
77
1H NMR (300 MHz, CDCl3) d=8.15 (2H, d, J=7.2 Hz,
References and Notes
H–Ph), 7.72 (2H, d, J=7.3 Hz, H–Ph), 7.61 (1H, m, H–
Ar), 7.54–7.35 (10H, m, H–Ph), 7.26 (1H, d, J=3.5 Hz,
H–Ar), 6.88 (1H, d, J=8.8 Hz, NH), 6.66 (1H, d, J=3.5
Hz, H–Ar), 6.30 (1H, s, H10), 6.28 (1H, d, J=9.7 Hz,
H13), 6.01 (1H, dd, J=2.6 Hz, 9.7 Hz, H30), 5.70 (1H,
d, J=7.2 Hz, H2), 5.44 (1H, d, J=2.6 Hz, H20), 5.17
(2H, s, Ar–CH2), 4.98 (1H, dd, J=9.7, 2.6 Hz, H5), 4.44
(1H, dd, J=11.4, 7.0 Hz, H7), 4.32 (1H, d, J=8.8 Hz,
H20a), 4.21 (1H, d, J=8.8 Hz, H20b), 3.82 (1H, d,
J=6.1 Hz, H3), 2.56 (1H, ddd, J=6.1, 9.7, 14.9 Hz,
H6a ), 2.47 (3H, s, 4-OAc), 2.49–2.35 (2H, m, H14a,
C7OH), 2.22 (1H, m, H14b), 2.24 (3H, s, 10-OAc), 1.93
(3H, s, H18), 1.83 (1H, m, H6b), 1.69 (3H, s, H19), 1.25
(3H, s, H16), 1.15 (3H, s, H17); FAB-MS, 1023
[M+H]+, 1045 [M+Na]+. Anal. (C53H54N2O19) calcd
C 62.23%, H 5.32%, N 2.74%, measured C 62.32%, H
5.64%, N 2.64%.
1. Brown, J. M. Cancer Res. 1999, 59, 5863. Brown, J. M.
Mol. Med. Today 2000, 6, 157.
2. Denny, W.A. In Cancer Chemotherapeutic Agents; Foye,
W.O. Ed., Am. Chem. Soc.: Washington DC, 1995; p 483.
3. Siim, B. G.; Denny, W. A.; Wilson, W. R. Oncol. Res. 1997,
9, 357.
4. Hay, M. P.; Wilson, W.; Denny, W. A. Tetrahedron 2000,
56, 645.
5. Brown, J. M. Br. J. Cancer 1993, 67, 1163.
6. Shyam, K.; Penketh, P. G.; Shapiro, M.; Belcourt, M. F.;
Loomis, R. H.; Rockwell, S.; Sartorelli, A. C. J. Med. Chem.
1999, 42, 941.
7. Kant, J.; Huang, S.; Wong, H.; Fairchild, C.; Vyas, D.;
Farina, V. Bioorg. Med. Chem. Lett. 1993, 3, 2471.
8. For mechanistic details, see: Carl, P. L.; Chakravarty, P. K.;
Katzenellenbogen, J. A. J. Med. Chem. 1981, 24, 479. Wak-
selman, M. New J. Chem. 1983, 7, 439. For recentreviews
about the prodrug concept, see: Sinhababa, D. K.; Thakker,
D. R. Adv. Drug Delivery Rev. 1996, 19, 241. Dubowchik,
G. M.; Walker, M.-A. Pharmacol. Ther. 1999, 83, 67. De
Groot, F. M. H.; Damen, E. W. P.; Scheeren, H. W. Curr.
Med. Chem. 2001, 18, 1093.
20-O-(5-Nitrothiophene-2-ylmethyloxycarbonyl)paclitaxel
(1i). Analogously to the preparation of 1b from 3i22 to
afford 1i as a white powder (105 mg, 94%); mp 158–
160 ꢀC; 1H NMR (300 MHz, CDCl3) d 8.15 (2H, d,
J=7.2 Hz, H–Ph), 7.79 (1H, d, J=3.9 Hz, H–Ar), 7.72
(2H, d, J=7.5 Hz, H–Ph), 7.61 (1H, m, H–Ph), 7.54–
7.35 (10H, m, H–Ph), 7.04 (1H, d, J=3.5 Hz, H–Ar),
6.86 (1H, d, J=9.2 Hz, NH), 6.66 (1H, d, J=3.5 Hz,
H–Ar), 6.30 (1H, s, H10), 6.27 (1H, d, J=9.2 Hz, H13),
6.01 (1H, dd, J=3.1Hz, 9.7 Hz, H30), 5.69 (1H, d,
J=7.0 Hz, H2), 5.46 (1H, d, J=2.6 Hz, H20), 5.27 (2H,
s, Ar–CH2), 4.98 (1H, dd, J=9.7, 1.7 Hz, H5), 4.45 (1H,
ddd, J=10.1, 6.6, 3.9 Hz, H7), 4.33 (1H, d, J=8.8 Hz,
H20a), 4.21 (1H, d, J=8.8 Hz, H20b), 3.82 (1H, d,
J=7.0 Hz, H3), 2.57 (1H, ddd, J=6.6, 9.7, 14.9 Hz,
H6a), 2.47 (3H, s, 4-OAc), 2.50–2.36 (2H, m, H14a,
C7OH), 2.24 (1H, m, H14b), 2.24 (3H, s, 10-OAc), 1.94
(3H, s, H18), 1.89 (1H, m, H6b), 1.69 (3H, s, H19), 1.24
(3H, s, H16), 1.14 (3H, s, H17); FAB-MS, 1039
[M+H]+, 1061[M+Na]+. Anal. (C53H54N2O18S) calcd
C 61.26%, H 5.24%, N 2.70%, S 3.09%, measured C
62.32%, H 5.64%, N 2.64%, S 2.36%.
9. Nicholls, D.; Gescher, A.; Griffin, R. J. Xenobiotica 1991,
21, 935.
10. Although reduction by endogenous thiols may hamper
the application of azides in hypoxia selective prodrugs.
Griffin, R. J.; Evers, E.; Davidson, R.; Gibson, A. E.;
Layton, D.; Irwin, W. J. J. Chem. Soc., Perkin Trans 1
1996, 1205.
11. Commercially available or prepared according to litera-
ture procedures: see ref 10. Beadle, C. D.; Bowman, W. R. J.
Chem. Res. Miniprint 1985, 5, 1814. Rao, V. P.; Jen, A. K.;
Wong, K. Y.; Drost, K. J. Tetrahedron Lett. 1993, 34, 1747.
´
12. Shiina, I.; Saitoh, K.; Frechard-Ortuno, I.; Mukaiyama,
T. Chem. Lett 1998, 3.
13. Damen, E. W. P.; Braamer, L.; Scheeren, J. W. Tetra-
hedron Lett. 1998, 39, 6081.
14. (a) Denis, J.-N.; Greene, A. E.; Serra, A. A.; Luche, M. J.
J. Org. Chem. 1986, 51, 46. (b) Kingston, D. G. I.; Chaudhary,
A. G.; Gunatilaka, A. A. L.; Middleton, M. L. Tetrahedron
Lett. 1994, 35, 4486.
15. The preparation of 20-carbamate prodrugs of paclitaxel
with an additional ring-closure spacer may lead to more stable
prodrugs, see: De Groot, F. M. H.; Van Berkom, L. W. A.;
Scheeren, J. W. J. Med. Chem. 2000, 43, 3093.
16. Wang, Z.; Kolb, H. C.; Sharpless, K. B. J. Org. Chem.
1994, 59, 5104.
In vitro cytotoxicity assay. The following human tumor
cell lines were used: MCF-7 and EVSA breastcarcinoma
cells, WIDE colon carcinoma cells, IGROV ovarian
carcinoma cells, M19 MEL melanoma cells, A498 renal
carcinoma cells and H226 non-small cell lung carcinoma
cells. The antiproliferative effects were determined apply-
ing the microculture sulforhodamine B (SRB) test23 and
are expressed as ID50 values. Results were averaged
from experiments that were performed in quadruplicate.
17. Sur, B.; Adak, M. M.; Pathak, T.; Hazra, B.; Banerjec, A.
Synthesis 1985, 653.
18. Mori, M.; Kawajiri, T.; Sayama, M.; Miyahara, T.;
Kozuka, H. Chem. Pharm. Bull. 1989, 37, 1904.
19. Showalter, H. D. H.; Sun, L.; Sercel, A. D.; Winters, R. T.;
Denny, W. A.; Palmer, B. D. J. Org. Chem. 1996, 61, 1155.
20. Adebayo, A. T. O. M.; Russel, B. W.; Salt, W. G. J.
Chem. Soc. Perkin Trans 1 1987, 2819.
21. Berry, J. M.; Watson, C. Y.; Threadgill, M. D. J. Chem.
Soc. Perkin Trans 1 1997, 1147.
Acknowledgements
22. Rao, V. P.; Jen, A. K.; Wong, K. Y.; Drost, K. J. Tetra-
hedron Lett. 1993, 34, 1747.
23. Kepers, Y. P.; Peters, G. J.; Van Ark-Otte, J.; Winograd,
B.; Pinedo, H. M. Eur. J. Cancer 1991, 27, 897.
We gratefully acknowledge the financial support from
Pharmachemie B.V. Haarlem and from The Academy
of Finland.