Synthesis of taxoids with improved cytotoxicity and solubility for use in tumor-specific delivery

Article abstract of DOI:10.1021/jm049705s

To develop effective taxane-antibody immunoconjugates, we have prepared a series of modified taxanes that have both improved toxicity and solubility in aqueous systems as compared to paclitaxel (1a). These taxanes have been modified at either the C-10 or

Full text of DOI:10.1021/jm049705s

4802
J . Med. Chem. 2004, 47, 4802-4805
Syn th esis of Ta xoid s w ith Im p r oved
Cytotoxicity a n d Solu bility for Use in
Tu m or -Sp ecific Deliver y
Michael L. Miller,* Elizabeth E. Roller, Robert Y.
Zhao, Barbara A. Leece, Olga Ab, Erkan Baloglu,
Victor S. Goldmacher, and Ravi V. J . Chari
ImmunoGen, Inc., 128 Sidney Street,
Cambridge, Massachussetts 02139
fied taxanes (i.e. 3) were found to be between 3 and
20-fold more potent than 1b against various tumor cell
lines.9 In light of this increased potency we became
interested in continuing to explore the SAR of these
disulfide-containing taxanes with the goal of de-
veloping highly cytotoxic taxanes with improved aque-
ous solubility.
Received April 20, 2004
Abstr a ct: To develop effective taxane-antibody immunocon-
jugates, we have prepared a series of modified taxanes that
have both improved toxicity and solubility in aqueous systems
as compared to paclitaxel (1a ). These taxanes have been
modified at either the C-10 or C-7 position and were found to
be very cytotoxic against both normal and multi-drug-resistant
(MDR) cells, as well as up to 30 times more soluble than
paclitaxel in various buffer systems.
To pursue these efforts, we became interested in
incorporating both heterocyclic and poly(ethylene glycol)
(PEG) substituents within the disulfide linker to explore
their effects on potency and solubility. Previously, it has
been shown that the introduction of a piperazinyl
substituent at the C-10 position had a dramatic effect
on the solubility of various taxanes.10 As a result of these
findings, we were interested in preparing taxoids in
which a piperazinyl ring was incorporated between the
C-10 position and the disulfide linker of taxanes such
as 2 and 3. As shown in Scheme 1, taxane 7 was pre-
pared through the coupling of the previously described
taxane 48 with p-nitrophenyl chloroformate followed by
the addition of piperazinyl disulfide 6 to give the desired
C-10 carbamate. Removal of the silyl protecting groups
with HF/pyridine gave the desired taxane in good
overall yield. In addition, to further explore the effects
on cytotoxicity of taxoids with a 3,5-dimethoxybenzoyl
substituent at C-2, we also prepared taxoid 8 using a
slightly different procedure. Thus, treatment of taxane
59 with carbonyl diimidazole (CDI) followed by the
addition of 6 and subsequent removal of the silyl
protecting groups gave 8 in good isolated yields.
In addition to looking into the incorporation of a
piperazinyl substituent at C-10, we were also interested
in preparing taxanes that possessed a small poly-
(ethylene glycol) (PEG) linker to enhance the solubil-
ity.11 In light of this, we prepared two small PEG linkers
containing either 4- or 10-ethyleneoxy (4-PEG or 10-
PEG) units that still had the desired disulfide moiety
present. The synthesis of the 4-PEG disulfide containing
carboxylic acid 12 is shown in Scheme 2. The synthesis
began with the treatment of tetraethylene glycol (9) with
tert-butyl acrylate in the presence of 1 mol % Na
followed by tosylation of the remaining free hydroxyl
group to give 10.12 Reaction of 10 with the potassium
salt of O-ethyl xanthic acid in ethanol gave xanthate
11 in 92% yield.13 Conversion to the free thiol was
accomplished with the use of hydroxylamine, followed
by the generation of the methyl disulfide using methyl
methanethiosulfonate in a phosphate buffer. Finally,
removal of the tert-butyl ester in the presence of TFA
and triethylsilane gave the desired free acid 12 in good
overall yield.
Recently, the potential for the use of monoclonal
antibodies in cancer therapy has been recognized with
the approvals of Rituxan,1 for lymphoma, Herceptin,2
for breast cancer and Erbitux,3 for colorectal cancer.
While these antibodies have proven their therapeutic
value, they are only moderately cytotoxic and therefore
are often more effective when used in combination with
other chemotherapeutic drugs. Nevertheless, a vast
majority of monoclonal antibodies are only weakly
potent and thus not useful as anticancer agents. How-
ever, the exquisite selectivity of these antibodies for
binding to tumor-associated antigens renders them
excellent vehicles for the targeted delivery of highly
cytotoxic small molecular weight drugs.4 Linkage of
cytotoxic drugs to monoclonal antibodies takes advan-
tage of the long in vivo half-life of the antibody (typically
several weeks for a humanized antibody). The drug
conjugate remains nontoxic during circulation and is
activated only upon binding to the tumor cell surface,
followed by internalization and release of the fully active
drug inside the target cell.
Paclitaxel (1a )5 and docetaxel (1b )6 are among the
most active anticancer agents in clinical use today, being
commonly used against ovarian cancer, breast cancer,
and non small cell lung cancer. Despite their contribu-
tion to chemotherapy, these taxanes suffer a number
of undesirable difficulties, such as the poor selectivity
for killing normal vs cancer cells, the development of
multidrug resistance (MDR), and a severe lack of sol-
ubility in aqueous systems. While the issue of selectivity
can potentially be addressed through the development
of an immunoconjugate selective for a tumor marker on
the surface of a cancer cell,7 it remains of interest to
develop taxanes that overcome the MDR phenotype
while possessing increased water solubility to facilitate
conjugation to antibodies in aqueous solutions.
Previously, we have described the synthesis of disul-
fide-containing taxanes such as 2 that were linked to
monoclonal antibodies generating an immunoconju-
gate.8 In addition, our initial structure-activity rela-
tionship (SAR) studies revealed that certain C-2 modi-
The 10-PEG linker 16 was prepared through a similar
path. Thus, treatment of hexaethylene glycol (13) with
10.1021/jm049705s CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/28/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4803
Sch em e 1a
Sch em e 4a
a
Reagents and conditions: (i) for 7, (a) Et3N, p-nitrophenyl
chloroformate, then 6, 71%. (b) HF/pyridine, CH3CN, 73%. For 8,
(a) CDI, imidazole, pyridine, 60 °C, then 6, 60 °C 14 h, 63%. (b)
HF/pyridine, CH3CN, 92%.
Sch em e 2a
a
Reagents and conditions: (i) (a) 5% HCl in ethanol, 79%. (b)
EDC (2 equiv), DMAP (1 equiv), acid a or b (1 equiv), a : 75%, b:
51%. (c) HF/pyridine, CH3CN, a : 83%, b: 88%. (ii) (a) DIC (3-10
equiv), DMAP (1-3 equiv), CH2Cl2 or toluene, acid b, or c (3-10
equiv), rt or 60 °C, b: 72%, c: 68%. (b) HF/pyridine, CH3CN, b:
74%, c: 67%.
a
Ta ble 1. In Vitro Cytotoxicity of Taxoids
Reagents and conditions: (i) (a) 1 mol % Na, tert-butyl acrylate,
20 h, 78%. (b) tosyl chloride, Et3N, DIC, CH3CN, 80%. (ii)
EtOCS2K, EtOH, rt, 4 h, 92%. (iii) (a) 0.5 M H2NOH‚HCl (pH )
7.5), EtOH/H2O. (b) CH3SO2SCH3, 1.0 M NaH2PO4 (pH ) 7.0),
EtOH, 50% two steps. (c) TFA, Et3SiH, CH2Cl2, 67%.
IC50, nMa
taxoid
A549b
3.0
MCF7c
paclitaxel, 1a d
1.7
1.0
1.0 ( 0.04
0.05 ( 0.016
1.0 ( 0.1
0.039 ( 0.015
0.073 ( 0.006
0.32 ( 0.004e
0.063 ( 0.015
0.58 ( 0.07e
docetaxel, 1bd
1.0
Sch em e 3a
2
3
7
8
17a
17b
18b
18c
0.80 ( 0.02
0.09 ( 0.01
1.10 ( 0.06
0.30 ( 0.02
0.13 ( 0.03
1.0 ( 0.05e
0.29 ( 0.02
1.1 ( 0.13e
a
The concentration of compound that inhibits 50% of the growth
of cancer cell line after 72 h of drug exposure and are expressed
a
b
Reagents and conditions: (i) Trityl chloride, pyridine, 74%.
as the mean ( SD based on three separate determinations. Non
(ii) (a) tBuO-K+, toluene, then 10, 67%. (b) H2, 10% Pd/C, MeOH,
77%. (c) Tosyl chloride, Et3N, DIC, CH3CN, 86%. (iii) (a) EtOCS2K,
EtOH, rt, 86%. (b) 0.5 M H2NOH‚HCl (pH ) 7.5), EtOH/H2O. (c)
CH3SO2SCH3, 1.0 M NaH2PO4 (pH ) 7.0), EtOH, 60% two steps.
(d) TFA, Et3SiH, CH2Cl2, 82%.
d
small cell lung carcinoma. c Human breast carcinoma. See ref
8. e IC50 based on two separate determinations.
desired C-7 4-PEG-modified taxoid. The C-10-modified
taxoids 18b,c were prepared by treating 5 with the
desired carboxylic acid (12 or 16) in the presence of DIC
and DMAP. Reaction with the 10-PEG acid 16 required
that the reaction be heated to 60 °C for an extended
period of time (2-5 days) in order for the reaction to go
to completion, but no decomposition of the reaction was
observed. Finally, removal of the protecting groups with
HF/pyridine gave taxoids 18b,c in good overall yield.
The biological activities of taxanes 7, 8, 17a ,b, and
18b,c were measured against the non small cell lung
carcinoma, A549, and the human breast carcinoma cell
line, MCF-7. As was seen with taxoids 2 and 3,9 the
effects of modification at C-2 were found to be critical
for achieving high potency. As shown in Table 1, taxoid
7, with a C-2 benzoyl group, was found to be very similar
in potency to parent taxoid 2. However, the C-2
dimethoxybenzoyl-modified taxoid 8 was found to be
significantly more potent, being some 25-fold more
potent against MCF7 cells.
trityl chloride gave the monoprotected glycol 14.14
Treatment of 14 with potassium tert-butoxide followed
by the addition of the tosylated PEG 10 gave the desired
10-PEG. Removal of the trityl protecing group with
hydrogen over Pd/C, followed by reaction with tosyl
chloride, gave 15 which was then easily converted to
the required 10-PEG disulfide linker 16 following the
same procedure described earlier for the conversion of
10 to 12.
With the desired PEG coupling units in hand, we next
turned our attention to the synthesis of taxoids 18b,c
as well as their C-7 modified counterparts 17a ,b. As
shown in Scheme 4, the approach to preparing taxane
17a began by treating 5 with 5% HCl in ethanol to give
the free hydroxyls at both the C-7 and C-10 positions.
Treatment of this intermediate with 1 equiv of meth-
yldithioproprionic acid in the presence of EDC (2 equiv)
and DMAP (1 equiv) resulted in selective acylation at
the C-7 position giving the desired ester in 75% isolated
yield. Removal of the silyl protecting groups with HF/
pyridine gave the desired taxoid 17a in good yield.
Taxoid 17b was prepared following an identical proce-
dure to that described for 17a using acid 12, giving the
Incorporation of the disulfide linker at the C-7 posi-
tion was also shown to be well tolerated toward potency,
as 17a was found to be between 6- and 13-fold more
potent than 2 against both cell lines as a result of the
C-2 modification. The results for the PEG-ylated taxoids

4804 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Letters
Ta ble 2. Solubility of Selected Taxoids
solubility (µg/mL)a
were found to be quite interesting. Modification of C-10
with either the 4-PEG (18b) or the 10-PEG (18c)
disulfide gave taxoids that showed high potency. It was
anticipated that as the size of the PEG was increased,
there would be a decrease in potency, and indeed the
10-PEG taxane 18c is about 10-fold less potent than the
corresponding non-PEGylated taxoid 3 against both cell
lines tested. Interestingly, the 4-PEG-containing taxane
18b shows only a slight loss in activity as compared to
3 but had the potential to be substantially more soluble
as a result of the presence of the short PEG linker.
Since the conjugation of cytotoxic drugs with antibod-
ies must be carried out in aqueous systems, only small
amounts of organic solvents may be used without an
effect on the antibody. Typically, suitable amounts of
organic cosolvents used vary from about 5 to 10%. To
test the enhancement in solubility for these modified
taxoids, we chose a system that contained 10% of an
organic solvent (DMA, DMSO, or ethanol) in the typical
conjugation media (0.1 M potassium phosphate buffer
with 1 mM EDTA (pH ) 6.5)). Thus, 200 µg of taxane
was dissolved in 50 µL of organic solvent and then
diluted to a volume of 500 µL with buffer. The mixture
was then vortexed and centrifuged, and the supernatant
was analyzed spectrophotometrically to determine the
concentration of taxane in solution.
As shown in Table 2, the solubility of paclitaxel in
these three solvent systems is very low, ranging between
4 and 8 µg/mL in all. The modified parent taxanes 3
and 17a were both found to have solubility that was
roughly the same as that which was observed for
paclitaxel. Taxoid 8 was found be from 4 to 8-fold more
soluble as a result of the piperazinyl group at C-10.
Interestingly, the taxanes in which the PEGs had been
incorporated showed very different results. Taxane 17b,
with the 4-PEG at C-7, was shown to gain only very
minimal advantages in solubility over 17a , being at best
only 6-fold more soluble. However, the taxanes modified
at C-10 were found to show a very impressive increase
in solubility as a result of these short PEG linkers. As
seen with 18b, the addition of the short 4-PEG linker
was found to increase the solubility approximately 10
to 14-fold over that of 1a . Taxoid 18c displayed the
highest degree of solubility with a 32-fold increase in
solubility over 1a in 10% EtOH; however, it’s loss in
potency makes it an unlikely candidate for further
development.
taxoid
10% DMA
10% DMSO
10% EtOH
paclitaxel, 1a
3
8
17a
17b
18b
18c
6
8
8
14
26
4
17
82
110
4
10
32
2
13
55
127
26
10
11
79
97
a
Solubility measurements are expressed as the mean of three
determinations. Standard deviations were generally found to be
less than ( 5% for all cases.
Ta ble 3. Cytotoxicity against Drug-Resistant Cell Lines
IC50 nMa
cell line
paclitaxel (1a )
3
18b
MCF7b
1
830
830
2.2
110
50
1.6
15
9.4
0.4
15
37.5
1
4
4
0.53
0.65
1.2
0.5
NCI ADR RESc
R/Sd
19
38
1.6
LCC6/P9e
LGM MDRf
R/Sd
18
11.25
0.45
0.9
2
Namalwag
Namalwa MDRh
R/Sd
a
The concentration of compound that inhibits 50% of the growth
b
of cancer cell line after 72 h of drug exposure. MCF7: human
breast carcinoma. c NCI ADR RES:MDR human breast carcinoma.
d
Ratio of activities, drug-resistant (R) vs drug-sensitive (S) cell
lines. e LCC6/P9: human breast carcinoma. f LGM MDR: MDR
g
h
human breast carcinoma. Namalwa:Burkitts lymphoma. Na-
malwa MDR: MDR Burkitts lymphoma.
LCC6/P9, and Namalwa) and their corresponding drug-
resistant cancer cell lines (NCI ADR RES, LGM MDR,
Namalwa MDR). As shown in Table 3, both taxanes
were found to be significantly more potent than pacli-
taxel against all three pairs of cell lines tested. In fact,
taxane 3 showed a greater than 50-fold increase in
potency against the drug-resistant breast cancer cell line
NCI ADR RES as compared to paclitaxel. It is interest-
ing to note that taxane 18b, despite the incorporated
4-PEG linker, maintains potency that is basically
equivalent to that of 3 against both drug-sensitive and
drug-resistant cell lines.
We have prepared a series of highly potent taxanes
modified at C-2 with a 2,5-dimethoxybenzoyl group. In
addition, these taxanes were found to be up to 50-fold
more potent than paclitaxel against various drug-
resistant cancer cell lines. With the additional incorpo-
ration of either heterocyclic or PEG linkers, these
taxoids were also found to be significantly more soluble
in an aqueous buffer system without compromising
potency. Taxane 18b was found to be highly potent and
up to 14 times more soluble than paclitaxel. Efforts to
further expand on the SAR of these taxanes, as well as
the development of taxane-antibody immunoconju-
gates, are currently underway and will be reported in
due course.
On the basis of these results, we decided to fur-
ther examine the cytotoxicity profile of taxoids 3 and
18b . It has previously been reported that a series of
C-2-modified taxoids were shown to be highly cyto-
toxic against both drug-sensitive and drug-resistant
cell lines.15 Therefore, we tested paclitaxel, 3 and
18b against three different cancer cell lines (MCF7,
Ack n ow led gm en t. The authors thank Dr. Wei
Zhang and Dr. Lintao Wang of ImmunoGen, Inc. for
obtaining HRMS data for all compounds.
Su p p or tin g In for m a tion Ava ila ble: Detailed synthetic
procedures and characterization data for compounds 7, 8, 12,

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4805
(8) Ojima, I.; Geng, X.; Wu, X.; Qu, C.; Borella, C. P.; Xie, H.;
Wilhelm, S. D.; Leece, B. A.; Bartle, L. M.; Goldmacher, V. S.;
Chari, R. V. J . Tumor-Specific Novel Taxoid-Monoclonal Anti-
body Conjugates. J . Med. Chem. 2002, 45, 5620-5623.
(9) Miller, M. L.; Roller, E. E.; Wu, X.; Leece, B. A.; Goldmacher, V.
S.; Chari, R. V. J .; Ojima, I. Synthesis of Potent Taxoids for
Tumor-Specific Delivery Using Monoclonal Antibodies. Bioorg.
Med Chem. Lett. 2004, 15, 4079-4082.
16, 17, 18, and their intermediates. This material is available
free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Newman, R. In Tumor Targeting in Cancer Therapy; Page, M.,
Ed.; 2002; pp 225-237.
(2) Leyland-J ones, B. Trastuzumab: Hopes and Realities. Lancet
Oncol. 2002, 3, 137-144.
(3) Reynolds, N. A.; Wagstaff, A. J . Cetuximab in the Treatment of
Metastatic Colorectal Cancer. Drugs 2004, 64, 109-118.
(4) Chari, R. V. J . Targeted Delivery of Chemotherapeutics: Tumor-
Activated Prodrug Therapy. Adv. Drug Delivery Rev. 1998, 31,
89-104.
(5) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A.
T. Plant Antitumor Agents. VI. Isolation and Structure of Taxol,
a Novel Antileukemic and Antitumor Agent From Taxus brevi-
folia. J . Am. Chem. Soc. 1971, 93, 2325-2327.
(6) Guenard, D.; Gueritte-Vogelein, F.; Potier, P. Taxol and Taxo-
tere: Discovery, Chemistry, and Structure-Activity Relation-
ships. Acc. Chem. Res. 1993, 26, 160-167.
(7) (a) Safavy, A.; Bonner, J . A.; Waksal, H. A.; Buchsbaum, D. J .;
Gillespie, G. Y.; Khazaeli, M. B.; Arani, R.; Chen, D. T.;
Carpenter, M.; Raisch, K. P. Synthesis and Biological Evaluation
of Paclitaxel-C225 Conjugate as a Model for Targeted Drug
Delivery. Bioconjugate Chem. 2003, 14, 302-310. (b) Guillemard,
V.; Saragovi, H. U. Taxane-Antibody Conjugates Afford Potent
Cytotoxicity, Enhanced Solubility, and Tumor Target Delivery.
Cancer Res. 2001, 61, 694-699. (c) Correa, J . J .; Page, M.
Synthesis and Evaluation of Paclitaxel Immunoconjugate With
Antitumor Activity In Vitro. Tumor Targeting in Cancer Therapy;
Page, M., Ed.; Humana Press: Totowa, NJ , 2002; pp 165-178.
(10) Abe, A.; Shimizu, H.; Sawada, S.; Ogawa, T.; Nagata, H. U.S.
Patent 6,136,808, 2000.
(11) (a) Greenwald, R. B.; Gilbert, C. W.; Pendri, A.; Conover, C. D.;
Xia, J .; Martinez, A. Drug Delivery Systems: Water Soluble
2′Poly(ethylene glycol) Ester Prodrugs-Design and in Vivo
Effectiveness. J . Med. Chem. 1996, 39, 424-431. (b) Greenwald,
R. B.; Pandri, A.; Bolikal, D. Highly Water Soluble Taxol
Derivatives: 7-Polyethylene Glycol Carbamates and Carbonates.
J . Org. Chem. 1995, 60, 331-336.
(12) Seitz, O.; Kunz. H. HYCRON, an Allylic Anchor for High-
Efficiency Solid-Phase Synthesis of Protected Peptides and
Glycopeptides. J . Org. Chem. 1997, 62, 813-826.
(13) Zalipsky, S.; Albericio, F.; Slomczynska, U.; Barany, G. A
Convenient General Method for Synthesis of Na- or Nw-
dithiasuccinoyl (Dts) Amino Acids and Dipeptides: Application
of Polyethylene Glycol as a Carrier for Functional Purification.
Int. J . Pept. Protein Res. 1987, 30, 740-783.
(14) Chen, Y.; Baker, G. L. Synthesis and Properties of ABA
Amphiphiles J . Org. Chem. 1999, 64, 6870-6873.
(15) Ojima, I.; Wang, T.; Miller, M. L.; Lin, S.; Borella, C. P.; Geng,
X.; Pera, P.; Bernacki, R. J . Synthesis and Structure-Activity
Relationships of New Second-Generation Taxoids. Bioorg. Med.
Chem. Lett. 1999, 9, 3423-3428.
J M049705S