Paclitaxel esters of malic acid as prodrugs with improved water solubility

Article abstract of DOI:10.1016/S0968-0896(99)00301-6

The synthesis of paclitaxel esters of malic acid is described. These compounds were found to have improved water solubility and are stable in solution at neutral pH. The C2' modified compounds behave as prodrugs, that is, paclitaxel is generated upon expo

Full text of DOI:10.1016/S0968-0896(99)00301-6

Bioorganic & Medicinal Chemistry 8 (2000) 427±432
Paclitaxel Esters of Malic Acid as Prodrugs with Improved
Water Solubility
Eric W. P. Damen, a Peter H. G. Wiegerinck, a Lesly Braamer, a Duncan Sperling, a
Dick de Vos b and Hans W. Scheeren a,*
aDepartment of Organic Chemistry, NSR Center for Molecular Structure, Design and Synthesis, University of Nijmegen, Toernooiveld,
6525 ED Nijmegen, The Netherlands
bPharmachemie B.V. Haarlem, Swensweg 5, Postbus 552, 2003 RN Haarlem, The Netherlands
Received 6 August 1999; accepted 8 November 1999
AbstractÐThe synthesis of paclitaxel esters of malic acid is described. These compounds were found to have improved water
solubility and are stable in solution at neutral pH. The C20 modi®ed compounds behave as prodrugs, that is, paclitaxel is generated
upon exposure to human plasma, whereas the C7 modi®ed derivatives do not. 20-Malyl paclitaxel sodium salt demonstrated
enhanced antitumour activity and less toxicity in a P388 murine leukaemia in vivo model when compared to paclitaxel. # 2000
Elsevier Science Ltd. All rights reserved.
Introduction
other prodrugs reported the solubilising moiety used is
known to be non-toxic, since it is an acid of the Krebs
cycle. We are aware that 20-succinyl paclitaxel and salts
thereof have already been published3,5 and that these
compounds have a slightly improved in vivo activity.
A major drawback is the instability of these compounds
in slightly basic aqueous solution which may very well
hamper the clinical application of these compounds.
This instability may be caused by assistance of the car-
boxylate in the hydrolysis of the 20-ester functionality.5
Our e€orts focus on malic acid derivatives of paclitaxel.
We rationalised that the additional hydroxyl function-
ality may improve stability by forming an intramole-
cular hydrogen bond between either carbonyl which
induces conformational rigidity, thereby retarding
hydrolysis. We realised that, for the same reason, this
might lead to a slightly diminished water solubility. For
preparing our target compounds it is obvious that the
anhydride strategy as described for the succinyl deriva-
tives cannot be used for malic acid. 20-Malyl paclitaxel
3 was prepared in excellent yield by condensing pacli-
taxel with protected malic acid18 in the presence of
dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP) at 10 ꢀC, followed by deprotection.
Upon addition of sodium hydrogen carbonate the
sodium salt 4 was obtained. The same reaction, carried
out with an excess of protected malic acid in re¯uxing
dichloromethane, a€orded 20,7-dimalyl paclitaxel 6
(Scheme 1).
Paclitaxel1 (Taxol1) 1 is a potent anticancer agent used
clinically to treat advanced ovarian, breast and non-
small cell lung cancer. Although paclitaxel has demon-
strated to be a unique antitumour agent it has several
disadvantages. One of the major problems is its poor
water solubility. Paclitaxel is administered in a vehicle
containing ethanol and Cremophor EL1, which is con-
sidered to cause some hypersensitivity reactions.2 In
addressing the solubility problems, many research
groups have reported syntheses and biological evalua-
tions of water soluble paclitaxel derivatives. These ana-
logues have polar substituents coupled to paclitaxel
either at the C20- or at the C7-hydroxyl group. The
solubilising moieties can be (salts of) carboxylic acids,3±5
phosphates,6±8 sulphonates,9 amines,10,11 sugar deriva-
tives,12,13 or polyethylene glycol.14±16 In most cases
these moieties are coupled to paclitaxel via an ester or
carbonate linkage.
Synthesis
We report the synthesis and biological evaluation of
malic acid17 paclitaxel derivatives. In contrast to many
*Corresponding author. Tel.: +31-24-65-2331; fax: +31-24-65-2929;
e-mail: jsch@sci.kun.nl
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00301-6

428
E. W. P. Damen et al. / Bioorg. Med. Chem. 8 (2000) 427±432
7-Malyl paclitaxel 9 was synthesised starting from bac-
catin III.19 This is the ®rst example of a semisynthetic
water soluble derivative, since it was not prepared from
paclitaxel itself. After reaction of baccatin III with pro-
tected malic acid as described above the side chain was
introduced using the oxazolidine method.20 Removal of
the protective groups resulted in the desired compound
(Scheme 2).
Biological Activity
All derivatives demonstrated improved water solubility
(20±60 times) when compared to paclitaxel and proved
to be stable when incubated in PBS bu€er (pH 7.4) at
37 ꢀC for 48 h, since no liberated paclitaxel was detected
(HPLC). In contrast to compounds 6 and 9, derivatives
3 and 4 generated paclitaxel when incubated in human
Scheme 1. Synthesis of derivatives 3, 4 and 6. Reagents: (i) (S)-1,2-O-isopropylidene-malic acid, DCC, DMAP, CH2Cl2, 10 ꢀC, 3 h, 99%; (ii)
HOAc:THF:water (1:1:1), r.t., 20 h, 94%; (iii) NaHCO3, acetone, water, 100%; (iv) (S)-1,2-O-isopropylidene-malic acid, DIC, DMAP,CH2Cl2,
re¯ux, 48 h, 72%. (v) HOAc:THF:water (1:1:1), 89%.
Scheme 2. Synthesis of 7-malyl paclitaxel. Reagents: (i) (S)-1,2-O-isopropylidene-malic acid, DCC, DMAP, CH2Cl2, r.t., 24 h, 86% at 81% con-
version; (ii) (4R,5S)-3-benzoyl-2,4-diphenyl-1,3-oxazolidine-5-carboxylic acid, DCC, DMAP, CH2Cl2, r.t., 3 h, 74%; (iii) HCl, EtOAc, r.t., 15 min,
89%.

E. W. P. Damen et al. / Bioorg. Med. Chem. 8 (2000) 427±432
429
plasma. Therefore 20-malyl paclitaxel and its sodium salt
act as prodrugs and the 7-malyl and 20,7-dimalyl deri-
vatives do not. Hydrolysis rate of sodium salt 4 in
human plasma is particularly encouraging. All com-
pounds were tested for cytotoxicity using de®ned human
tumour cell lines. The results are summarised in Table 1.
salt 4 was found to be much more active than paclitaxel
in the in vivo p388 tumour model on the basis of a
higher therapeutic index than paclitaxel and the pre-
sence of long term survivors.
Experimental
Derivatives 6 and 9, bearing a 7-malyl group, were sig-
ni®cantly less active than paclitaxel. Compounds 3 and
4 showed similar activity when compared to paclitaxel.
General methods
1,2-O-Isopropylidene-malic acid,18 baccatin III,19 and
the protected paclitaxel side chain ((4R,5S)-3-benzoyl-
2,4-diphenyl-1,3-oxazolane-5-carboxylic acid20 were
prepared as described in literature. Dichloromethane
was distilled from calcium hydride prior to use. All
other materials were obtained from commercial sources
and used without further puri®cation. Paclitaxel was a
generous gift from Pharmachemie B.V. Haarlem.
Prodrug 4 was subjected to in vivo evaluation against
the murine leukaemia P388 tumour. The maximum tol-
erated dose (MTD) was established at approximately
100 mg/kg once i.p. (MTD of paclitaxel: 50 mg/kg once
ip). From this MTD the doses for the therapeutic experi-
ments were derived. The results are depicted in Table 2.
Compound 4 caused a dose-dependent increase of
survival time. The optimum e€ect was obtained with
25 mg/kg/inj in a qd 1±4 schedule. This e€ect was sig-
ni®cantly higher than the best result with the reference
paclitaxel (T/C 204), which is higher than the range T/C
118±194 reported in literature.21,22 In the paclitaxel test
there were no 30-days survivors, whereas compound 4
showed 2/6 45-days survivors.
NMR spectra were recorded on a Bruker AC300
(300 MHz) or a Bruker AM400 (400 MHz) spectro-
meter. Chemical shifts are given in ppm (d) relative to
TMS as internal standard. The purity of the prodrugs
was checked with RP-HPLC. All compounds were at
least 90% pure, with some small impurities present,
which were integrated as less than 2%.
In summary, we have synthesised in good yields some
novel water soluble paclitaxel derivatives and have
demonstrated that 20-malyl paclitaxel 3 and its sodium
salt 4 act as prodrugs. Most interestingly, in addition to
the excellent water solubility, 20-malyl paclitaxel sodium
HPLC: Rheodyne injection valve (20 mL loop); Lichro-
spher 5RP18 column (200Â3 mM, Chrompack); UV-
detector (Model 759A, Applied Biosystems); eluent:
CH3CN:MeOH:H2O: 5:1:4 in 10 mM NH4OAc (pH
5.0). The detection of the (pro)drugs was performed at
Table 1. Water solubility, stability and cytotoxicity of malic acid paclitaxel derivatives
a
T1/2 (h)
IC50b (ng/mL)
Compd
Water solubility
(mg/mL)
PH 7.4
Plasma
MCF7
EVSA-T
WIDR
IGROV
M19
A498
Paclitaxel
0.01
0.2
0.6
0.5
0.3
Ð
>24
No pacl.c
No pacl.c
No pacl.c
Ð
20
4
<3
<3
<3
69
<3
<3
<3
59
<3
<3
<3
167
589
10
<3
233
49
<3
3
6
311
1344
<3
39
13
436
1485
3
4
6
9
No pacl.c
No pacl.c
390
300
241
aHalf-live values (T1/2), time in which 50% of the derivative is degraded to paclitaxel.
bIC50, drug concentration required to inhibit cell proliferation to 50% versus untreated cells (37 ꢀC, 72 h).
cNo paclitaxel detected (HPLC).
Table 2. In vivo test results for prodrug 4 against a murine P388 tumour model
Compd
Mice
Treatment
(days)
Dose
(mg/kg/inj.)
Toxic deaths
(days)
BWCa (%)
(day 1±8)
Mean survival
(days)‹SDd
T/Cc (%)
45 days
survivor
Saline
4
4
4
4
6
6
6
6
6
6
6
6
1±4
1±4
1±4
1±4
1
1
1
1±4
9
15
6b
22
10b
12
16
8
9.2‹0.4
11.2‹4.7
32.5‹12.3
9.7‹0.5
12.8‹4.6
11.3‹1.0
10.8‹1.5
18.8‹4.8
0/6
0/6
2/6
0/6
0/6
0/6
0/6
0/6
50
25
12.5
100
75
1 (2)
1 (6)
122
353
105
139
123
117
204
4
4
Paclitaxel
50
12.5
aBody weight loss relative to control.
bThe relative increase in body weight is due to ascites development.
cTreated over control.
dStandard deviation.

430
E. W. P. Damen et al. / Bioorg. Med. Chem. 8 (2000) 427±432
226 nM, where it is supposed that the extinction coe-
cients of paclitaxel and paclitaxel prodrugs are equal.
The concentrations were determined by measuring the
relative area of paclitaxel or the paclitaxel prodrugs.
20-Malyl paclitaxel sodium salt (4). To a solution of 20-
malyl paclitaxel (3) (250.9 mg, 0.259 mmol) in acetone
(10 mL) was added a solution of NaHCO3 (21.7 mg,
0.259 mmol) in demineralised water (20 mL). The reac-
tion mixture was stirred at room temperature for 1 h.
Sodium salt 4 (256 mg, 0.259 mmol, 100%) was isolated
after removal of the acetone in vacuo and freeze drying.
TLC analysis was performed on Merck precoated silica
gel 60 F-254 plates. Spots were visualised with UV light
and a 10% H2SO4 solution (1 L) containing ammonium
molybdate (25 g) and ceric ammonium sulfate (10 g)
followed by charring. Column chromatography was
carried out on Merck Kieselgel 60.
mp 195 ꢀC; H NMR (300 MHz, CDCl3): in accordance
1
with the structure of compound 3; FABMS, 992
[M+H]+, 1014 [M+Na]+.
20,7-Bis-(1,2-O-isopropylidene-malyl)-paclitaxel (5).
A
Mass spectra were obtained with a double focusing VG
7070E spectrometer.
solution of paclitaxel (50 mg, 0.059 mmol) and 1,2-O-
isopropylidene-malic acid (51 mg, 0.29 mmol) in CH2Cl2
(5 mL) was stirred at 0 ꢀC. Next, diisopropylcarbodii-
mide (DIC) (100 mL, 0.64 mmol) and DMAP (7.5 mg,
0.061 mmol) were added. After 1 h, the mixture was
heated to re¯ux temperature and stirred for 3 days. The
mixture was ®ltered over Hy¯o. The ®ltrate was diluted
with CH2Cl2 (30 mL) and washed with a saturated
NaHCO3 solution, demineralised water, 0.5 N KHSO4
solution and brine. The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was
puri®ed via column chromatography (EtOAc:hexane,
1:1), yielding 5 (49 mg, 0.0421 mmol, 72%). mp 139 ꢀC;
1H NMR (400 MHz, CDCl3): d 8.14 (2H, d, J=7.6 Hz,
H-Ph), 7.80 (2H, d, J=7.6 Hz, H-Ph), 7.61 (1H, t,
J=7.4 Hz, H-Ph), 7.39 (10H, m, H-Ph), 7.02 (1H, d,
20-1,2-O-Isopropylidene-malyl) paclitaxel (2). A solution
of paclitaxel (853 mg, 1.00 mmol) and 1,2-O-iso-
propylidene-malic acid (191 mg, 1.10 mmol) in CH2Cl2
(25 mL) was stirred at 10 ꢀC. Next, DCC (227 mg,
1.10 mmol) and DMAP (15 mg, 0.12 mmol) were added.
After stirring for 3 h at 10 ꢀC, the mixture was ®ltered
over Hy¯o, diluted with EtOAc (75 mL) and washed
with a saturated NaHCO3 solution, demineralised water
and brine, dried over Na2SO4 and concentrated in
vacuo. The residue was puri®ed via column chromato-
graphy (EtOAc:hexane, 1:1), yielding
2 (999 mg,
0.99 mmol, 99%). mp 138 ꢀC; 1H NMR (300 MHz,
CDCl3): d 8.15 (2H, d, J=7.2 Hz, H-Ph), 7.80 (2H, d,
J=7.4 Hz, H-Ph), 7.61 (1H, t, J=7.2 Hz, H-Ph), 7.45
0
JNH-3 =9.2 Hz, NH), 6.24 (1H, s, H10), 6.23 (1H, m,
0
5.69 (1H, d, J2-3=6.9 Hz, H2), 5.66 (1H, m, H7), 5.55
0
(10H, m, H-Ph), 7.01 (1H, d, JNH-3 =9.2 Hz, NH), 6.29
0
H13), 5.99 (1H, dd, J3 -NH=9.2 Hz, J3 -2 =3.0 Hz, H3 ),
0
0
0
(1H, s, H10), 6.26 (1H, m, H13), 6.00 (1H, dd, J3 -NH
=
9.2 Hz, J3 -2 =3.0 Hz, H3 ), 5.69 (1H, d, J2-3=7.1 Hz,
0
H2), 5.52 (1H, d, J2 -3 =3.0 Hz, H2 ), 4.97 (1H, d, J5-6
0
4.84 (1H, m, CH-malyl), 4.64 (1H, m, CH-malyl), 4.33
(1H, d, J20a-20b=8.4 Hz, H20a), 4.19 (1H, d, J20b-20a=
0
0
0
(1H, d, J2 -3 =3.0 Hz, H2 ), 4.97 (1H, d, J5-6=9.4, H5),
0
0
8.0, H5), 4.44 (1H, m, H7), 4.32 (1H, d, J20a-20b
0
0
=
=
8.4 Hz, H20a), 4.21 (1H, d, J20b-20a=8.4 Hz, H20b),
4.12 (1H, m, CH-malyl), 3.82 (1H, d, J3-2=7.1 Hz,
H3), 2.96 (2H, m, CH2-malyl), 2.51 (1H, m, H6), 2.46
(3H, s, OCOCH3), 2.36 (1H, m, H14a), 2.23 (3H, s,
OCOCH3), 2.19 (1H, m, H14b), 2.03 (1H, m, H6), 1.93
(1H, s, H18), 1.69 (3H, s, H16), 1.57 (3H, s, acetonide),
1.51 (3H, s, acetonide), 1.25 (3H, s, H17), 1.13 (3H, s,
H19); FABMS, 1010 [M+H]+, 1032 [M+Na]+.
8.4 Hz, H20b), 3.94 (1H, d, J3-2=6.9 Hz, H3), 3.10
(2H, m, CH2-malyl), 2.97 (2H, m, CH2-malyl), 2.60
(1H, m, H6), 2.45 (3H, s, OCOCH3), 2.35 (1H, m,
H14a), 2.25 (1H, m, H14b), 2.21 (3H, s, OCOCH3), 1.97
(1H, s, H18), 1.85 (1H, m, H6), 1.81 (3H, s, H16),
1.58 (6H, s, acetonide), 1.56 (6H, s, acetonide), 1.21
(3H, s, H17), 1.16 (3H, s, H19); FABMS, 1166
[M+H]+.
20-Malyl paclitaxel (3). Compound 2 (500 mg, 0.496 mmol)
20,7-Bis-(malyl) paclitaxel (6). Compound 5 (40 mg,
0.0343 mmol) was dissolved in a mixture of HOAc:
THF:H2O (4:1:2 mL). The mixture was stirred at 45 ꢀC
for 6 h. Next, the organic solvents were removed by
evaporation in vacuo. The residue was diluted by water
(50 mL) and freeze dried, yielding 5 (33 mg, 0.0304mmol,
was dissolved in
a mixture of HOAc:THF:H2O
(25:25:25 mL). The mixture was stirred at room tem-
perature for 6 h. Next, the organic solvents were
removed by evaporation in vacuo. The residue was
diluted by water (50 mL) and freeze dried, yielding 2
(451 mg, 0.466 mmol, 94%). Mp 148±151 ꢀC. H NMR
89%). mp 166±168 ꢀC; H NMR (400 MHz, CDCl3): d
1
1
(300 MHz, CDCl3): d 8.16 (2H, d, J=7.6 Hz, H-Ph),
7.93 (2H, d, J=7.6 Hz, H-Ph), 7.61 (1H, t, J=7.3 Hz,
H-Ph), 7.36 (11H, m, H-Ph and NH), 6.30 (1H, s, H10),
8.11 (2H, d, J=7.6 Hz, H-Ph), 7.84 (2H, d, J=7.5 Hz,
H-Ph), 7.63 (1H, t, J=7.5Hz, H-Ph), 7.37 (11H, m, H-Ph
and NH), 6.25 (1H, s, H10), 6.16 (1H, m, H13), 5.99
0
d, J2-3=6.4 Hz, H2), 5.66 (1H, m, H7), 5.63 (1H, d,
0
0
0
0
(1H, dd, J3 -NH=8.8 Hz, J3 -2 =2.9 Hz, H3 ), 5.67 (1H,
0
0
6.28 (1H, m, H13), 6.08 (1H, dd, J3 -NH=9.2 Hz, J3 -2
=
2.8 Hz, H30), 5.68 0(1H, d, J2-3=7.3 Hz, H2), 5.51 (1H, d,
0
0
0
0
0
J2 -3 =2.8 Hz, H2 ), 4.99 (1H, d, J5-6=8.0, H5), 4.46
(1H, m, H7), 4.33 (1H, d, J20a-20b=8.4 Hz, H20a), 4.27
(1H, m, CH-malyl), 4.22 (1H, d, J20b-20a=8.4 Hz, H20b),
3.82 (1H, d, J3-2=7.3 Hz, H3), 3.03 (2H, m, CH2-malyl),
2.55 (1H, m, H6), 2.53 (3H, s, OCOCH3), 2.40 (1H, m,
H14a), 2.23 (3H, s, OCOCH3), 2.13 (1H, m, H14b), 1.93
(1H, s, H18), 1.88 (1H, m, H6), 1.69 (3H, s, H16), 1.21
(3H, s, H17), 1.14 (3H, s, H19); FABMS, 992 [M+Na]+.
J2 -3 =2.8 Hz, H2 ), 4.94 (1H, d, J5-6=7.6, H5), 4.48
(2H, m, CH-malyl), 4.32 (1H, d, J20a-20b=7.9 Hz,
H20a), 4.17 (1H, d, J20b-20a=7.9 Hz, H20b), 3.88 (1H, d,
J3-2= 6.4 Hz, H3), 2.97 (4H, m, CH2-malyl), 2.51 (1H,
m, H6), 2.44 (3H, s, OCOCH3), 2.36 (2H, m, H14), 2.07
(3H, s, OCOCH3), 1.93 (1H, s, H18), 1.86 (1H, m, H6),
1.79 (3H, s, H16), 1.20 (3H, s, H17), 1.18 (3H, s, H19);
FABMS, 1108 [M+Na]+.

E. W. P. Damen et al. / Bioorg. Med. Chem. 8 (2000) 427±432
431
7-(1,2-O-Isopropylidene-malyl) baccatin III (7). To a
solution of baccatin III (107 mg, 0.183 mmol) and 1,2-
O-isopropylidene-malic acid (43 mg, 0.274 mmol) in
CH2Cl2 (5 mL) were added DCC (60 mg, 0.292 mmol)
and DMAP (7.5 mg, 0.06 mmol). After stirring for 20 h
another portion of 1,2-O-isopropylidene-malic acid
(43 mg, 0.274 mmol) and DCC (60 mg, 0.292 mmol)
were added. After stirring for another 4 h still some
starting material was present, but also the formation of
a di€erent compound (possibly 7,13-bis-(1,2-O-iso-
propylidene-malyl) baccatin III) was observed on TLC.
The mixture was diluted with EtOAc (25 mL) and
washed with a saturated NaHCO3 solution. The organic
layer was washed with water, dried over Na2SO4 and
concentrated in vacuo. The residue was puri®ed via
column chromatography (EtOAc:hexane, 1:1), yielding
7-Malyl paclitaxel (9). Compound 8 (74 mg, 0.0.067 mmol)
was dissolved in EtOAc (5 mL). Next, an 3.5 N HCl
solution in EtOAc (1 mL) was added. The mixture was
stirred at room temperature for 15 min. The solvents
were removed by evaporation in vacuo. The residue was
diluted by water (50 mL) and freeze dried, yielding 9
(64 mg, 0.059 mmol, 89%). Mp 166±168 ꢀC; H NMR
1
(400 MHz, CDCl3): d 8.11 (2H, d, J=7.6 Hz, H-Ph),
7.84 (2H, d, J=7.5 Hz, H-Ph), 7.63 (1H, t, J=7.5 Hz,
H-Ph), 7.37 (11H, m, H-Ph and NH), 6.25 (1H, s, H10),
0
0
0
6.16 (1H, m, H13), 5.99 (1H, dd, J3 -NH=8.8 Hz, J3 -2 =
2.9 Hz, H30), 5.67 (1H, d, J2-3=6.40Hz, H2), 5.66 (1H,
0
m, H7), 5.63 (1H, d, J2 -3 =2.8 Hz, H2 ), 4.94 (1H, d, J5-6
0
=
=
7.6, H5), 4.48 (2H, m, CH-malyl), 4.32 (1H, d, J20a-20b
7.9 Hz, H20a), 4.17 (1H, d, J20b-20a=7.9 Hz, H20b), 3.88
(1H, d, J3-2=6.4 Hz, H3), 2.97 (4H, m, CH2-malyl), 2.51
(1H, m, H6), 2.44 (3H, s, OCOCH3), 2.36 (2H, m, H14),
2.07 (3H, s, OCOCH3), 1.93 (1H, s, H18), 1.86 (1H, m,
H6), 1.79 (3H, s, H16), 1.20 (3H, s, H17), 1.18 (3H, s,
H19); FABMS, 1108 [M+Na]+.
baccatin III (20 mg 0.034 mmol) and
7 (88 mg,
0.121 mmol, 66%). Mp 160±163 ꢀC; 1H NMR
(300 MHz, CDCl3): d 8.10 (2H, d, J=7.2 Hz, H-Ph),
7.57 (1H, t, J=7.2 Hz, H-Ph), 7.47 (2H, d, J=7.2 Hz,
H-Ph), 6.26 (1H, s, H10), 5.67 (2H, m, H2, H7), 4.91
(4H, m, H13 H5 CH-malyl H20), 4.20 (1H, d, J20a-20b
=
Biological activity
8.4 Hz, H20a), 4.13 (1H, d, J20b-20a=8.4 Hz, H20b),
3.95 (1H, d, J3-2=7.1 Hz, H3), 2.92 (1H, dd, Jgem
=
Water solubility.23 Paclitaxel and paclitaxel deriva-
tives were suspended in water or PBS-bu€er (pH 7.4)
until a concentration was reached of 2 mg/mL. The sus-
pensions were sonicated for 15 min and centrifuged
(13,000 g) for 10 min. The above ¯uid was analysed,
using HPLC.
17.2 Hz, JCH-malyl=3.0 Hz, CH2a-malyl), 2.56 (1H, dd,
Jgem=17.2 Hz, JCH-malyl=9.0 Hz, CH2b-malyl), 2.51
(1H, m, H6a), 2.46 (3H, s, OCOCH3), 2.36 (1H, m,
H14a), 2.23 (3H, s, OCOCH3), 2.19 (1H, m, H14b), 2.03
(1H, m, H6b), 1.93 (1H, s, H18), 1.69 (3H, s, H16), 1.60
(3H, s, acetonide), 1.56 (3H, s, acetonide), 1.21 (3H, s,
H17), 1.08 (3H, s, H19); FABMS, 744 [M+H]+, 766
[M+Na]+.
Stability in human plasma and PBS-bu€er. The pacli-
taxel prodrugs (3, 4, 6 and 9) were dissolved in water,
sonicated and centrifuged. A 100 mL of the above ¯uid was
mixed with 400 mL of plasma (heparin) or PBS-bu€er
(pH 7.4). The solutions were incubated at 37 ꢀC and on
di€erent points in time (T=0, 0.5, 1, 4, 20, 48 h) 50 mL
was extracted with 150 mL of EtOAc. After mixing for
2 min (using a vortex), this mixture was centrifuged
(2 min, 13,000 g) and 100 mL EtOAc was evaporated
(30 min, in vacuo). The (pro)drugs were dissolved in
50 mL eluent and analysed by HPLC.
20,30-O,N-((4-methoxy)benzylidene-7-1,2-O-isopropylidene-
malyl) paclitaxel (8).
A
solution of
7
(68 mg,
0.094 mmol) and (4R,5S)-3-benzoyl-2,4-diphenyl-1,3-
oxazolidine-5-carboxylic acid (57 mg, 0.141 mmol) in
CH2Cl2 (5 mL) was stirred at 0 ꢀC. Next, DCC (31 mg,
0.150 mmol) and a few crystals of DMAP were added.
After stirring for 8 h at room temperature, the mixture
was diluted with EtOAc (25 mL) and washed with a
saturated NaHCO3 solution, demineralised water, 0.5 N
KHSO4 solution and brine. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The residue
was puri®ed via column chromatography (EtOAc:
hexane, 1:1), yielding 8 (77 mg, 0.692 mmol, 74%). Mp
In vitro cytotoxicity assay. The compounds under study
were dissolved to a concentration of 177147 ng/mL in
5% DMSO in full RPMI growth medium. On day 0,
200 mL of trypsinised tumour cells (2Â103 cells/well)
were plated in 96-wells ¯atbottom microtiter plates
(Costar, no. 3799, Badhoevedorp, The Netherlands).
The plates were preincubated 24 h at 37 ꢀC, 5% CO2 to
allow the cells to adhere.
128±130 ꢀC; H NMR (300 MHz, CDCl3): d 8.04 (2H,
1
d, J=7.2 Hz, H-Ph), 7.60 (1H, t, J=7.2 Hz, H-Ph),
7.46 (3H, m, H-Ph), 7.35 (3H, m, H-Ph), 7.30 (8H, m,
H-Ph), 7.91 (1H, d, J=8.6 Hz, H-Ph-OMe), 6.85 (1H, s,
oxazolidine) 6.31 (1H, t, J13-14=8.8, H13), 6.26 (1H, s,
H10), 5.66 (2H, m, H2, H7), 5.55 (1H, br s, H30), 4.85
On day 2, 100 mL of the highest drug concentration was
added to the wells of column 12 and from there diluted
3-fold to column 3 by serial transfer of 100 mL using an
eight channel micropipette. The ®nal volume of column
3 was adjusted to 200 mL with PBS. Column 2 was used
for the blank. To column 1 PBS was added to diminish
interfering evaporation.
(3H, m, H5 CH-malyl H20), 4.20 (1H, d, J20a-20b
=
8.4 Hz, H20a), 4.13 (1H, d, J20b-20a=8.4 Hz, H20b), 3.95
(1H, d, J3-2=7.1 Hz, H3), 3.85 (3H, s, Ph-OMe) 3.09
(1H, dd, Jgem=17.2 Hz, JCH-malyl=3.0 Hz, CH2a-malyl),
2.62 (1H, dd, Jgem=17.2 Hz, JCH-malyl=9.0 Hz, CH2b-
malyl), 2.51 (1H, m, H6a), 2.46 (3H, s, OCOCH3), 2.36
(1H, m, H14a), 2.23 (3H, s, OCOCH3), 2.19 (1H, m,
H14b), 2.03 (1H, m, H6b), 1.93 (1H, s, H18), 1.69 (3H,
s, H16), 1.60 (3H, s, acetonide), 1.57 (3H, s, acetonide),
1.25 (3H, s, H17), 1.13 (3H, s, H19); FABMS, 1151
[M+Na]+.
On day 7 the incubation was terminated by washing the
plates twice with PBS. Subsequently the cells were ®xed
with 10% trichloroacetic acid in Milli Q water (Millipore,
Etten Leur, The Netherlands) and placed at 4 ꢀC for 1 h.

432
E. W. P. Damen et al. / Bioorg. Med. Chem. 8 (2000) 427±432
After ®ve washings with tap water, the cells were stained
for at least 15 min with 0.4% SRB, dissolved in 1%
acetic acid, and subsequently washed with 1% acetic
acid to remove the unbound stain. The plates were air
dried and the bound protein was dissolved by using
150 mL 10 mmol/L tris base. The absorbance was read at
540 nM using an automatic microplate reader (Titertec,
Flow Laboratories Ltd., Irvine, UK). Data were used
for construction of concentration±response curves and
determination of the IC50-value (for more information
on the test methodology, see refs 24±26).
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Approximative toxicity. Two mice received 25, 50 100,
and 200 mg/kg of compound 4 once intraperitonealy
(ip). Mice treated with 200 mg/kg died 3 days later. The
substance caused a dose-dependent body weight reduc-
tion. The maximum tolerated dose (MTD) is approx-
imative 100 mg/kg once ip. From this dose dosages in
the therapeutic experiments were derived.
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In vivo P388 leukaemia screen. Paclitaxel and 20-malyl
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aqueous solutions. Female B6D2F1 mice were implan-
ted i.p. with P388 cells (106 cells/mouse) on day 0. The
mice were then treated ip with paclitaxel or the prodrug
for 4 consecutive days (for dose see Table 2). Control
groups received a saline solution. Medial survival times
of compound-treated (T) mice were compared to the
medial survival time of the control (C) mice. The ratio
of the two values for each compound treated group of
mice was multiplied by 100 and expressed as a per-
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Fichtner.27
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Acknowledgement
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27. For information on experimental details and test panel
background, see refs 21 and 22.
We gratefully acknowledge the ®nancial support from
Pharmachemie B.V. Haarlem.
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