Synthesis and protein binding of (4-carboxybutyl)carbamoyl- substituted taxoids

Article abstract of DOI:10.1002/1522-2675(20010711)84:7<1989::AID-HLCA1989>3.0.CO;2-M

(4-Carboxybutyl)carbamates 5 and 6, as well as 10, derived from 10-O-deacetylbaccatin III (1) and paclitaxel (2), respectively, were synthesized by reaction of unprotected 1 and 2′-O-(methoxyacetyl)paclitaxel (8), respectively, with trimethylsilyl 5-isocyanatopentanoate in good yields. The carbamoyl-taxoids were conjugated to bovine-serum albumin and analyzed by MALDI-TOF mass spectrometry.

Full text of DOI:10.1002/1522-2675(20010711)84:7<1989::AID-HLCA1989>3.0.CO;2-M

Helvetica Chimica Acta ± Vol. 84 (2001)
1989
Synthesis and Protein Binding of (4-Carboxybutyl)carbamoyl-
Substituted Taxoids
by Torsten Blitzkea)1), Alexander Baranovskyb)2), and Bernd Schneider*b)
a) Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle
b) Max-Planck-Institute for Chemical Ecology, Carl-Zeiss-Promenade 10, D-07745 Jena
(e-mail: schneider@ice.mpg.de)
(4-Carboxybutyl)carbamates 5 and 6, as well as 10, derived from 10-O-deacetylbaccatin III (1) and
paclitaxel (2), respectively, were synthesized by reaction of unprotected 1 and 2'-O-(methoxyacetyl)paclitaxel
(8), respectively, with trimethylsilyl 5-isocyanatopentanoate in good yields. The carbamoyl-taxoids were
conjugated to bovine-serum albumin and analyzed by MALDI-TOF mass spectrometry.
Introduction. ± Since the discovery and development of paclitaxel (Taxolꢀ) as an
important anticancer drug [1], a number of attemps have been made to synthesize
taxoids carrying potential spacer moieties to conjugate these compounds to proteins for
use as haptens in antibody preparation, to attach to fluorescent groups, or to improve
their water solubility by binding to hydrophilic molecules. For example, succinoyl-
taxoids of 10-O-deacetylbaccatin III (1) [2] and paclitaxel (2) [3][4] were prepared,
bound to bovine-serum albumine, and employed in the preparation of antibodies and
the development of immunoenzymatic assays. The 2'-O-glutarylpaclitaxel served as an
intermediate in the synthesis of a hexanediamine derivative, which was treated with
fluorescein isothiocyanate as a fluorescent dye [5]. Further taxoid derivatives
possessing a free amino group, e.g., 7-O-(l-alanyl)taxol [6], were also used to enable
coupling to fluorescent [7] or luminescent components [8]. Formation of carbamates is
considered another possibility to functionalize taxoid molecules. The (trichloroethoxy)-
carbonyl derivative (TROC) of paclitaxel (2) has been treated with primary amines to
prepare simple carbamates [9]. Polyethylene glycol conjugates at the 7-hydroxy group
of paclitaxel linked via a carbamate functionality were prepared from an isocyanate
precursor [10]. A carbamate side chain has been attached to the 10-O-position of 10-
deacetyl-7-O-(triethylsilyl)baccatin III by reaction with phenyl isocyanate or dime-
thylcarbamic chloride [11]. Isocyanates carrying a protected w-carboxy group should
be suitable to couple directly to reactive 7- and 10-hydroxy groups of 10-O-
deacetylbaccatin III (DAB; 1) or paclitaxel. In this paper, we report the synthesis of
taxoid carbamates of that type and demonstrate proper binding to bovine-serum
albumin.
1
)
)
Present address: Bell, Flavors and Fragrances, Schimmelstr. 1, D-04205 Miltitz.
Present address: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich
Str., Minsk, Belarus.
2

1990
Helvetica Chimica Acta ± Vol. 84 (2001)
Results and Discussion. ± Regioselective substitution of 10-O-deacetylbaccatin III
(1) at the 7- or 10-OH group requires selective protection of the 10- or 7-OH group,
respectively, generation of the lithium alkoxide, reaction with an electrophile, and
finally deprotection [11]. Since we were interested in both 7- and 10-O-substituted
derivatives, a non-regioselective approach including subsequent separation of the
product mixture seemed a reasonable alternative. Therefore, unprotected 10-O-
deacetylbaccatin III (1) was employed in the reaction with trimethylsilyl 5-isocyana-
topentanoate (4; Scheme 1). Starting material 4 was prepared from hexanedioic acid
via its anhydride 3 and reaction of the latter with azidotrimethylsilane [12]. In
comparison with literature data [12], the yield of anhydride 3 was significantly
improved to 78%.
Scheme 1
O
O
Me3SiN3
O
O
OCN
O SiMe3
- N2
3
4
As expected, the reaction of 1 with the isocyanato ester 4 in the presence of
dibutyltin dilaurate as a catalyst yielded three products: 7-O-[(4-carboxybutyl)carba-
moyl]-10-O-deacetylbaccatin III (5), 10-O-[(4-carboxybutyl)carbamoyl]-10-O-deace-
tylbaccatin III (6), and 7,10-bis-O-[(4-carboxybutyl)carbamoyl]-10-O-deacetylbaccatin
III (7; Scheme 2). The ratio of monocarbamates 5 and 6, and dicarbamate 7 was
determined by HPLC. The best yield of 5/6 and minimum amounts of the undesired
dicarbamate 7 were obtained after 16 h reaction at room temperature. Separation of
the regioisomers 5 and 6, and dicarbamate 7 was carried out by prep. HPLC. Mass and
NMR specroscopic data (see Exper. Part) confirmed their structures.
Scheme 2
3'
COOH
6'
HOOC
HN
NH
1'
HO
O
HO
O
O
O
O
OH
OH
O
O
10
7
4, SnBu2Lau2
7
+
+
1
THF, r.t., 16h
O
O
O
HO
H
HO
H
HO
H
HO
PhCOO
HO
PhCOO
HO
PhCOO
OAc
OAc
OAc
1
5
6
‡
Both carbamates 5 and 6 showed very similar MS and the same M at m/z 687. The 1H- and 13C-NMR
spectra exhibited additional signals of the (4-carboxybutyl)carbamoyl side chains. The chemical-shift difference
for H C(7) of the parent DAB (1) (d 4.27) and the corresponding carbamate 5 (d 5.46) of ca. 1.2 ppm indicated
attachment of the carbamate unit at C(7) in compound 5. A similar shift difference of 1 ppm was observed for
H
C(10) of 6 (d 6.32) in comparison with that of 1 (d 5.32). Attachment of the carbamoyl side chain was
further confirmed by long-rang connectivities between H C(7) and OCONH (157.5) of 5 and H C(10) and
OCONH (158.1) of 7 in the HMBC spectra.

Helvetica Chimica Acta ± Vol. 84 (2001)
1991
A similar procedure as for the DAB carbamates 5 and 6 was applied to synthesize
paclitaxel carbamate 10 (Scheme 3). Paclitaxel (2) was protected at the 2'-OH group by
methoxyacetyl (MeOCOCH2), a protection group, which has proved useful in taxoid
chemistry [13][14]. Reaction of 2'-O-(methoxyacetyl)paclitaxel (8) with trimethylsilyl
5-isocyanatopentanoate (4) yielded 2'-O-(methoxyacetyl)paclitaxel carbamate 9.
Separation by means of reversed-phase HPLC (gradient B, see Exper. Part) revealed
two products: a minor one (ca. 20%) at tR 9.7 min and the major component (ca. 80%)
1
at tR 14.2 min. H-NMR Analysis indicated the major component was the expected
compound 9 and the minor one the 2'-O-deprotected paclitaxel carbamate 10.
Complete deprotection under basic conditions smoothly gave the desired product 10,
which was purified by prep. HPLC. Mass spectra, 1D and heterocorrelated 2D NMR
data confirmed the structure of intermediates 8 and 9, and of the final product 10,
allowing complete assignment of the 1H- and 13C-NMR chemical shifts.
Scheme 3
3''
6''
HOOC
NH
1''
AcO
O
OH
AcO
O
O
O
O
O
10
1
7
4
4, SnBu2Lau2
3
NH
O
NH
O
13
2'
THF, r.t., 16h
O
O
H
O
O
H
2
HO
HO
O
OAc
OR
O
OAc
OR
O
O
1
9 R = MeOCH2CO
10 R = H
2 R = H
8 R = MeOCH2CO
Attachment of the carbamate moiety caused a downfield shift of H C(7) from d 4.40 in paclitaxel (2) or d
4.42 in 2'-O-(methoxyacetyl)paclitaxel (8) to d 5.50 in 9 and d 5.47 in the final product 10. A cross-peak between
C(7) and OCONH in the HMBC spectrum confirmed attachment of the carbamate unit to C(7). 13C-
H
Chemical-shift differences up to ca. 4 ppm were observed for the phenylisoserine side-chain C-atoms C(1') to
C(3') of 10 in comparison to compound 9 and, surprisingly, up to ca. 2.5 ppm for the terminal C-atoms (C(6'')
and COOH) of the carbamate side chain. The latter chemical-shift differences may be caused by changes in the
environment of the (4-carboxybutyl)carbamate unit: due to the cavity of the paclitaxel skeleton, this unit is
located spatially close to the MeOCH2CO group at the phenylisoserine side chain of 9.
Conjugation of the carbamate derivatives 5 and 6 to bovine-serum albumin (BSA)
as a carrier protein was achieved by the carbodiimide method [15]. MALDI-TOF Mass
spectrometry was used to determine the number of [(4-carboxybutyl)carbamoyl]DAB
units attached to BSA (m/z 66504). The average mass of the 7-O-[(4-carboxybutyl)-
carbamoyl]DAB (5)±BSA complex was determined as m/z 72958, indicating attach-
ment of 9.4 units of 5 to BSA. The m/z 72348 of 10-O-[(4-carboxybutyl)carbamoyl]-
DAB (6)±BSA complex was due to a 8.5 :1 ratio of 6/BSA. The average mass of 7-O-
[(4-carboxybutyl)carbamoyl]paclitaxel (10)±BSA was m/z 71741, corresponding to a
4.9 :1 ratio of 10/BSA. Antigens obtained by this procedure are suitable for the
preparation of antibodies against DAB and paclitaxel, respectively.

1992
Helvetica Chimica Acta ± Vol. 84 (2001)
Experimental Part
1. General. The 10-O-deacetylbaccatin III (DAB; 1) was a gift of P. Potier, Gif-sur-Yvette, France.
Paclitaxel (2), bovine-serum albumine (BSA), and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC)
were purchased from Sigma. Other chemicals and solvents were commercial products from Aldrich and Merck.
All reactions were carried out under a continuous stream of N2. Anal. HPLC: LiChrospherꢀ-100-RP-18 column
1
(250 Â 4 mm); linear gradient 30 ! 40% MeCN/H2O within 25 min, 0.6 ml min (gradient A); 45% MeCN/
1
H2O for 5 min, then to 70% MeCN/H2O within 25 min, 0.8 ml min (gradient B); detection by UV-DAD at
210 ± 400 nm. Prep. HPLC: Macherey-Nagel Nucleosilꢀ-100-7C18 column (250 Â 21 mm); linear gradient 20 !
50% MeCN/H2O within 30 min, 10.0 ml min 1; UV detection at 230 nm. NMR Spectra: 1H-NMR, 1H,1H-COSY,
HMBC, and HMQC, Bruker Avance DRX 500 with a 2.5-mm inverse-detection microprobe head; 13C-NMR
and DEPT, 2.5-mm broadband-detection microprobe head; 1H and 13C routine spectra, Varian Gemini 2000
300BB; CDCl3 or CD3OD as solvent and Me4Si as an internal standard; d in ppm, J in Hz. ESI-MS: TSQ
7000 Finnigan (electrospray voltage 4.5 kV; heated capillary, temp. 2208; sheath gas N2) coupled with a Micro-
Tech-Ultra-Plus-MicroLC system (reversed-phase C18 colum (4 mm, 1 Â 100 mm, Ultrasep); gradient H2O/
MeCN 4 :1 (each containing 0.2% AcOH) ! 1:9 within 15 min, then 1:9 for 10 min; flow rate 70 ml min 1).
HR-MS and/or combustion analysis data are not available. The compounds were used up completely in the
preparation of BSA conjugates. MALDI-TOF-MS of the conjugates: Micromass-TofSpec-2E time-of-flight
mass spectrometer operating in the linear mode (positive-ion detection); delayed ion extraction mode
measurements to enhance resolution; matrix, sinapic acid.
2. Starting Materials. Hexanedioic Anhydride (ˆOxepane-2,7-dione; 3). A mixture of hexanedioic acid (5 g,
34 mmol) and freshly distilled Ac2O (20 ml) was stirred under reflux for 12 h. AcOH and excess of Ac2O were
evaporated. The crude product was heated to 2208 for 10 min and purified by distillation at 84 ± 868/0.5 Torr:
3.4 g (78%) of 3 ([12]: 23%).
5-Isocyanatopentanoic Acid Trimethylsilyl Ester (4). Azidotrimethylsilane (3 ml, 20 mmol) was added
dropwise to a soln. of freshly prepared 3 (2 g, 16 mmol) in dioxane (12 ml) under continuous stirring at r.t. [12].
The mixture was slowly heated to 758 and, after N2 evolution had ceased, refluxed for additional 20 min.
Evaporation and distillation at 80 ± 838/0.3 Torr gave 4 (2.97 g, 87%). Moisture- and air-sensitive colorless oil. IR
(CHCl3): 2256 (NˆCˆO), 1711 (CˆO). 1H-NMR (300 MHz, CDCl3): 1.61 (m, CH2CH2); 2.30 (t, J ˆ 6.8,
CH2); 3.28 (t, J ˆ 6.3, CH2). 13C-NMR (75.5 MHz, CDCl3): 21.9 (CH2); 30.5 (CH2); 35.0 (CH2); 42.6 (CH2);
122.0 (NCO); 173.6 (COOR).
3. 10-O-Deacetylbaccatin III Carbamates. General Procedure. An excess of isocyanate 4 (400 ml) and
dibutyltin dilaurate (ˆdibutylbis(1-oxododecyloxy)stannane; 10 ml, 17 mmol) were added dropwise to a soln. of
10-O-deacetylbaccatin III (1; 30 mg, 55 mmol) in dry THF (5 ml). The mixture was stirred at r.t. for 16 h. Then
the reaction was quenched by the addition of MeOH (2 ml), thereby removing the Me3Si group. The solvent was
evaporated and the crude mixture diluted with MeCN/H2O 60 :40. After prepurification (RP18 cartridge,
isocratic MeCN/H2O 60 :40), the isomers were separated by prep. HPLC.
7-O-[(4-Carboxybutyl)carbamoyl]-10-O-deacetylbaccatin III (ˆ 5-{{{[(2aR,4S,4aS,6R,9S,11S,12S,12aR,
12bS)-12b-(Acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-6,9,11-trihydroxy-4a,
8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxet-4-yl]oxy}carbonyl}amino}pentanoic
Acid; 5). 1H-NMR (500 MHz, CD3OD): 5.62 (d, J ˆ 7.0, H C(2)); 4.08 (d, J ˆ 7.0, H C(3)); 2.28 (s, Ac); 5.01
(br. d, J ˆ 9.1, H C(5)); 2.50 (m, Ha C(6)); 1.85 (m, Hb C(6)); 5.46 (dd, J ˆ 10.3, 7.5, H C(7)); 3.06
(m, CH2(3')); 1.50 (m, CH2(4')); 1.60 (m, CH2(5')); 2.27 (m, CH2(6')); 5.53 (s, H C(10)); 4.80 (t, J ˆ 8.8,
H
C(13)); 2.38 (dd, J ˆ 15.6, 7.0, Ha C(14)); 2.25 (m, Hb C(14)); 1.08 (s, Me(16)); 1.07 (s, Me(17)); 2.08
(s, Me(18)); 1.78 (s, Me(19)); 4.22 (d, J ˆ 11.3, 1 H, CH2(20)); 4.20 (d, J ˆ 11.3, 1 H, CH2(20)); 8.12 (d, J ˆ 8.0,
2 Ho); 7.52 (dd, J ˆ 8.0, 7.6, 2 Hm); 7.63 (t, J ˆ 7.6, Hp). 13C-NMR (125 MHz, CD3OD): 79.4 (C(1)); 76.4 (C(2));
48.3 (C(3)); 81.7 (C(4)); 172.1 (MeCO); 22.7 (MeCO), 85.5 (C(5)); 34.8 (C(6)); 73.8 (C(7)); 157.5 (C(1')); 41.5
(C(3')); 30.4 (C(4')); 23.9 (C(5')); 36.2 (C(6')); 178.9 (C(7')); 57.6 (C(8)); 211.7 (C(9)); 76.3 (C(10)); 135.7
(C(11)); 145.1 (C(12)); 68.2 (C(13)); 40.6 (C(14)); 44.0 (C(15)); 20.8 (C(16)); 27.1 (C(17)); 15.4 (C(18)); 11.4
(C(19)); 77.6 (C(20)); 167.8 (PhCO); 131.6 (Cipso); 131.2 (Co); 129.7 (Cm); 134.5 (Cp). ESI-MS: 710 (100, [M ‡
‡
Na] ), 118 (30).
10-O-[(4-Carboxybutyl)carbamoyl]-10-O-deacetylbaccatin III (ˆ 5-{{{[(2aR,4S,4aS,6R,9S,11S,12S,
12aR,12bS)-12b-(Acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,9,11-trihydroxy-
4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxet-6-yl]oxy}carbonyl}amino}penta-
noic Acid; 6). 1H-NMR (500 MHz, CD3OD): 5.62 (d, J ˆ 6.90, H C(2)); 3.93 (d, J ˆ 6.90, H C(3)); 2.27
(s, Ac); 5.02 (br. d, J ˆ 8.7, H C(5)); 2.47 (m, Ha C(6)); 1.78 (m, Hb C(6)); 4.40 (dd, J ˆ 10.9, 6.9, H C(7));

Helvetica Chimica Acta ± Vol. 84 (2001)
1993
6.32 (s, H C(10)); 3.16 (m, CH2(3')); 1.56 (m, CH2(4')); 1.66 (m, CH2(5')); 2.32 (m, CH2(6')); 4.80 (t, J ˆ 9.0,
H
C(13)); 2.38 (dd, J ˆ 15.5, 7.7, Ha C(14)); 2.26 (m, Hb C(14)); 1.12 (s, Me(16)); 1.07 (s, Me(17)); 2.07
(s, Me(18)); 1.64 (s, Me(19)); 4.20 (d, J ˆ 10.9, 1 H, CH2(20)); 4.18 (d, J ˆ 10.9, 1 H, CH2(20)); 8.12 (d, J ˆ 7.9,
2 Ho); 7.51 (dd, J ˆ 7.9, 7.4, 2 Hm); 7.63 (t, J ˆ 7.4, Hp). 13C-NMR (125 MHz, CD3OD): 79.5 (C(1)); 76.4 (C(2));
48.3 (C(3)); 82.0 (C(4)); 172.1 (MeCO); 22.7 (MeCO); 86.0 (C(5)); 37.3 (C(6)); 72.9 (C(7)); 158.1 (C(1')); 41.6
(C(3')); 30.4 (C(4')); 23.3 (C(5')); 35.3 (C(6')); 176.4 (C(7')); 59.5 (C(8)); 207.5 (C(9)); 77.9 (C(10)); 133.3
(C(11)); 147.9 (C(12)); 68.2 (C(13)); 40.6 (C(14)); 44.0 (C(15)); 21.7 (C(16)); 27.2 (C(17)); 15.6 (C(18)); 10.2
(C(19)); 77.5 (C(20)); 167.8 (PhCO); 131.6 (Cipso); 131.1 (Co); 129.7 (Cm); 134.5 (Cp). ESI-MS: 710 (34, [M ‡
‡
Na] ), 118 (100).
7,10-Bis-O-[(4-carboxybutyl)carbamoyl]-10-O-deacetylbaccatin III (ˆ 5,5'-{[(2aR,4S,4aS,6R,9S,11S,
12S,12aR,12bS)-12b-(Acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-9,11-dihy-
droxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxet-4,6-diyl]bis(oxycarbonyl-
imino)}bis[pentanoic Acid]; 7). 1H-NMR (500 MHz, CD3OD): 5.63 (d, J ˆ 6.9, H C(2)); 4.04 (d, J ˆ 6.9,
H
C(3)); 2.28 (s, Ac); 5.02 (br. d, J ˆ 9.1, H C(5)); 2.60 (m, Ha C(6)); 1.76 (m, Hb C(6)); 5.48 (dd, J ˆ 10.0,
7.5, H C(7)); 3.11 (m, CH2(3'), CH2(3'')); 1.53 (m, CH2(4'), CH2(4'')); 1.60 (m, CH2(5'), CH2(5'')); 2.30
(m, CH2(6'), CH2(6'')); 6.39 (s, H C(10)); 4.80 (t, J ˆ 8.8, H C(13)); 2.37 (dd, J ˆ 15.8, 7.1, Ha C(14)); 2.25
(m, Hb C(14)); 1.13 (s, Me(16)); 1.06 (s, Me(17)); 2.13 (s, Me(18)); 1.75 (s, Me(19)); 4.21 (d, J ˆ 8.3, 1 H,
CH2(20)); 4.18 (d, J ˆ 8.3, 1 H, CH2(20)); 8.12 (d, J ˆ 8.0, 2 Ho); 7.52 (dd, J ˆ 8.0, 7.6, 2 Hm); 7.63 (t, J ˆ 7.6, Hp).
13C-NMR (125 MHz, CD3OD): 79.3 (C(1)); 76.1 (C(2)); 48.9 (C(3)); 81.8 (C(4)); 172.0 (MeCO); 22.7 (MeCO);
85.6 (C(5)); 34.8 (C(6)); 73.7 (C(7)); 157.3, 157.5 (C(1'), C(1'')); 41.7, 41.6 (C(3'), C(3'')); 30.1, 30.6 (C(4'),
C(4'')); 23.6, 23.5 (C(5'), C(5'')); 35.3, 35.2 (C(6'), C(6'')); 178.4, 178.3 (C(7'), C(7'')); 57.6 (C(8)); 206.7 (C(9));
77.8 (C(10)); 133.1 (C(11)); 147.1 (C(12)); 68.2 (C(13)); 40.4 (C(14)); 44.1 (C(15)); 21.3 (C(16)); 27.0 (C(17));
15.6 (C(18)); 11.4 (C(19)); 77.5 (C(20)); 167.8 (PhCO); 131.6 (Cipso); 131.1 (Co); 129.7 (Cm); 134.5 (Cp). ESI-MS:
‡
853 (100, [M ‡ Na] ), 118 (20).
4. Paclitaxel Carbamates. 2'-O-(Methoxyacetyl)paclitaxel (ˆ(aR,bS)-b-(Benzoylamino)-a-[(methoxyace-
tyl)oxy]benzenepropanoic Acid (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-Bis(acetyloxy)-12-(benzoyloxy)-
2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cy-
clodeca[3,4]benz[1,2-b]oxet-9-yl Ester; 8). Protection of paclitaxel (2) was carried out according to Greenwald
and co-workers [10]. The crude product was purified by prep. reversed-phase HPLC (63% yield after HPLC).
1H-NMR (500 MHz, CDCl3): 5.67 (d, J ˆ 7.1, H C(2)); 3.79 (d, J ˆ 7.1, H C(3)); 2.45 (s, AcO C(4)); 4.96
(br. d, J ˆ 9.3, H C(5)); 2.52 (m, Ha C(6)); 1.87 (m, Hb C(6)); 4.42 (dd, J ˆ 10.3, 6.6, H C(7)); 6.27
(s, H C(10)); 2.21 (s, AcO C(10)); 6.25 (t, J ˆ 8.6, H C(13)); 2.37 (dd, J ˆ 9.3, 15.4, Ha C(14)); 1.87
(m, Hb C(14)); 1.12 (s, Me(16)); 1.21 (s, Me(17)); 1.92 (s, Me(18)); 1.66 (s, Me(19)); 4.15 (d, J ˆ 16.6, 1 H,
CH2(20)); 4.08 (d, J ˆ 16.6, 1 H, CH2(20)); 5.58 (d, J ˆ 3.0, H C(2')); 4.30 (d, J ˆ 8.6, 1 H, MeOCH2CO); 4.18
(d, J ˆ 8.6, 1 H, MeOCH2CO); 3.37 (s, MeOCH2CO); 6.01 (dd, J ˆ 9.3, 3.0, H C(3')); 6.96 (d, J ˆ 9.3,
NH C(3')); 8.13 (d, J ˆ 7.8, 2 Ho of Ph(1)); 7.58 (t, J ˆ 7.5, Hp of Ph(1)); 7.50 (m, 2 Hm of Ph(1), Hp of Ph
(3)); 7.42 ± 7.32 (m, 2 Ho of Ph(2), 2 Hm of Ph(3), 2 Hm of Ph(2), Hp of Ph(2)); 7.71 (d, J ˆ 7.8, 2 Ho of Ph(3)).
‡
‡
ESI-MS: 949 ([M ‡ Na] , 3), 926 (100, M ).
7-O-[(4-Carboxybutyl)carbamoyl]-2'-O-(methoxyacetyl)paclitaxel (ˆ(aR,bS)-b-(Benzoylamino)-a-
[(methoxyacetyl)oxy]benzenepropanoic Acid (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-Bis(acetyloxy)-12-
(benzoyloxy)-4-{{[(4-carboxybutyl)amino]carbonyl}oxy}-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-11-hy-
droxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxet-9-yl Ester; 9). To a soln. of
8 (20 mg, 22 mmol) in dry THF (5 ml) under N2 4 (48 ml) and dibutyltin dilaurate (5 ml, 8.4 mmol) were added
dropwise. The mixture was stirred at r.t. for 12 h. Then, MeOH (2 ml) was added, the mixture evaporated, and
the residue diluted with MeCN/H2O 60 :40. After prepurification (RP 18 cartridge, isocratic MeCN/H2O
1
60 :40), the final purification was carried out by prep. HPLC. H-NMR (500 MHz, CD3OD): 5.65 (d, J ˆ 7.0,
H
C(2)); 3.95 (d, J ˆ 7.0, H C(3)); 2.44 (s, AcO C(4)); 5.01 (br. d, J ˆ 9.2, H C(5)); 2.56 (m, Ha C(6));
1.80 (m, Hb C(6)); 5.50 (dd, J ˆ 10.1, 7.0, H C(7)); 3.09 (m, CH2(3'')); 1.52 (m, CH2(4'')); 1.61 (m, CH2(5''));
2.31 (m, CH2(6'')); 6.47 (s, H C(10)); 2.12 (s, AcO C(10)); 6.11 (t, J ˆ 9.0, H C(13)); 2.27 (m, Ha C(14));
1.95 (m, Hb C(14)); 1.14 (s, Me(16)); 1.15 (s, Me(17)); 1.99 (s, Me(18)); 1.77 (s, Me(19)); 4.19 (s, CH2(20)); 5.63
(d, J ˆ 5.6, H C(2')); 4.21 (m, MeOCH2CO); 3.37 (s, MeOCH2CO); 5.93 (d, J ˆ 5.6, H C(3')); 8.13 (d, J ˆ 8.0,
2 Ho of Ph(1)); 7.58 (dd, J ˆ 8.0, 7.4, 2 Hm of Ph(1)); 7.67 (t, J ˆ 7.4, Hp of Ph(1)); 7.50 (d, J ˆ 8.0, 2 Ho of Ph(2));
7.45 (dd, J ˆ 8.0, 7.4, 2 Hm of Ph(2)); 7.30 (t, J ˆ 7.4, Hp of Ph(2)); 7.81 (d, J ˆ 8.0, 2 Ho of Ph(3)); 7.45 (dd, J ˆ 8.0,
7.4, 2 Hm of Ph(3)); 7.54 (t, J ˆ 7.4, 1 Hp of Ph(3)). 13C-NMR (125 MHz, CD3OD): 79.0 (C(1)); 76.0 (C(2)); 48.3
(C(3)); 82.0 (C(4)); 171.7 (MeCO C(4)); 23.3 (MeCO C(4)); 85.5 (C(5)); 34.8 (C(6)); 73.3 (C(7)); 157.6
(C(1'')); 41.6 (C(3'')); 30.0 (C(4'')); 23.4 (C(5'')); 34.7 (C(6'')); 177.6 (C(7'')); 57.6 (C(8)); 204.4 (C(9)); 76.8

1994
Helvetica Chimica Acta ± Vol. 84 (2001)
(C(10)); 170.3 (MeCO C(10)); 20.7 (MeCO C(10)); 134.7 (C(11)); 142.1 (C(12)); 73.2 (C(13)); 36.5 (C(14));
44.7 (C(15)); 22.2 (C(16)); 26.9 (C(17)); 15.0 (C(18)); 11.9 (C(19)); 77.4 (C(20)); 170.1 (C(1')); 76.1 (C(2'));
171.3 (MeOCH2CO); 70.3 (MeOCH2CO); 59.8 (MeOCH2CO); 55.1 (C(3')); 167.7 (Ph(1)CO); 131.4 (Cipso of
Ph(1)); 131.3 (Co of Ph(1)); 130.2 (Cm of Ph(1)); 134.6 (Cp of Ph(1)); 135.6 (Cipso of Ph(2)); 128.5 (Co of Ph(2));
128.8 (Cm of Ph(2)); 129.7 (Cp of Ph(2)); 170.7 (Ph(3)CO); 138.3 (Cipso of Ph(3)); 128.7 (Co of Ph(3)); 129.6 (Cm
‡
‡
of Ph(3)); 133.0 (Cp of Ph(3)). ESI-MS: 1091 (4, [M ‡ Na] ), 1069 (12, [M ‡ H] ), 1009 (40), 562 (38), 228
(100).
7-O-[(4-Carboxybutyl)carbamoyl]paclitaxel (ˆ(aR,bS)-b-(Benzoylamino)benzenepropanoic Acid
(2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-Bis(acetyloxy)-12-(benzoyloxy)-4-{{[(4-carboxybutyl)amino]car-
bonyl}oxy}-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-11-hydroxy-4a,8,13,13-tetramethyl-5-oxo-7,11-meth-
ano-1H-cyclodeca[3,4]benz[1,2-b]oxet-9-yl Ester; 10). Deprotection of 9 was done under basic conditions [16].
In a typical experiment, to a soln. of 9 (1.84 mg, 1.72 mmol) in MeOH (1.5 ml), 5% aq. NH3 soln. (0.2 ml, ca.
0.59 mmol) was added. The resulting soln. was stirred at r.t. for 1 h and then evaporated without heating. The
obtained residue was chromatographed by reversed-phase HPLC (isocratic MeCN/H2O 45 :55): 10 (1.32 mg,
77%). With iPr2EtN instead of NH3 as base, a similar result was obtained. 1H-NMR (500 MHz, CD3OD): 5.64
(d, J ˆ 6.9, H C(2)); 3.92 (d, J ˆ 6.9, H C(3)); 2.35 (s, AcO C(4)); 4.99 (br. d, J ˆ 9.5, H C(5)); 2.53
(m, Ha C(6)); 1.79 (m, Hb C(6)); 5.47 (dd, J ˆ 10.3, 6.6, H C(7)); 3.08 (t, J ˆ 6.6, CH2(3'')); 1.51 (m,
CH2(4'')); 1.60 (m, CH2(5'')); 2.24 (m, CH2(6'')); 6.46 (s, H C(10)); 2.12 (s, AcO C(10)); 6.15 (dt, J ˆ 9.0, 1.1,
H
C(13)); 2.23 (m, Ha C(14)); 2.00 (ddd, J ˆ 14.9, 9.0, 1.1, Hb C(14)); 1.13 (s, Me(16)); 1.15 (s, Me(17)); 1.96
(s, Me(18)); 1.76 (s, Me(19)); 4.19 (s, CH2(20)); 4.75 (d, J ˆ 5.3, H C(2')); 5.65 (d, J ˆ 5.3, H C(3')); 8.11
(d, J ˆ 8.0, 2 Ho of Ph(1)); 7.57 (dd, J ˆ 8.0, 7.4, 2 Hm of Ph(1)); 7.67 (t, J ˆ 7.4, Hp of Ph(1)); 7.49 (d, J ˆ 8.0, 2 Ho
of Ph(2)); 7.42 (dd, J ˆ 8.0, 7.4, 2 Hm of Ph(2)); 7.29 (t, J ˆ 7.4, Hp of Ph(2)); 7.85 (d, J ˆ 8.0, 2 Ho of Ph(3)); 7.46
(dd, J ˆ 8.0, 7.4, 2 Hm of Ph(3)); 7.54 (t, J ˆ 7.4, Hp of Ph(3)). 13C-NMR (125 MHz, CD3OD; starred signals may
be interchanged): 79.0 (C(1)); 76.0 (C(2)); 48.3 (C(3)); 82.1 (C(4)); 171.8 (MeCO C(4)); 23.2 (MeCO C(4));
85.5 (C(5)); 34.8 (C(6)); 73.2 (C(7)); 157.4 (C(1'')); 41.8 (C(3'')); 30.2 (C(4'')); 24.3 (C(5'')); 36.9 (C(6'')); 180.2
(C(7'')); 57.6 (C(8)); 204.3 (C(9)); 76.8 (C(10)); 170.3 (MeCO C(10)); 20.7 (MeCO C(10)); 134.7 (C(11));
142.0 (C(12)); 72.3 (C(13)); 36.6 (C(14)); 44.7 (C(15)); 22.1 (C(16)); 26.8 (C(17)); 14.8 (C(18)); 11.9 (C(19));
77.4 (C(20)); 174.5 (C(1')); 74.8 (C(2')); 57.7 (C(3')); 167.7 (Ph(1)CO); 131.4 (Cipso of Ph(1)); 131.3 (Co of Ph(1));
129.6 (Cm of Ph(1)); 134.7 (Cp of Ph(1)); 135.7 (Cipso of Ph(2)); 129.7 (Co of Ph(2)); 128.7* (Cm of Ph(2)); 129.1
(Cp of Ph(2)); 170.4 (Ph(3)CO); 140.0 (Cipso of Ph(3)); 128.6* (Co of Ph(3)); 129.6* (Cm of Ph(3)); 132.9 (Cp of
‡
‡
Ph(3)). ESI-MS: 1019 (100, [M ‡ Na] ), 997 (48, [M ‡ H] ), 752 (21).
5. BSA Conjugation. DAB±BSA Conjugate. To a soln. of 5 or 6 (7.5 mg, ca. 11 mmol) in pyridine/H2O 1:1
(1 ml), a soln. of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC; 10 mg) in pyridine/H2O 1:1
(0.5 ml) was added dropwise within 3 min while stirring. After 10 min at r.t., bovine-serum albumin (BSA;
16.6 mg) in H2O (1 ml) was added dropwise within 3 min while stirring. The mixture was further stirred for 20 h
at r.t. Dialysis for 4 d against H2O followed by lyophilization yielded the BSA conjugates of compounds 5 and 6,
resp. MALDI-TOF-MS: 72958 (5-BSA), 72348 (6-BSA); reference, 66504 (BSA).
Paclitaxel±BSA Conjugate. According to the same procedure, with 10 (3.1 mg, ca. 3.1 mmol) in pyridine/
H2O 1:1 (0.4 ml), EDAC (4.1 mg) in pyridine/H2O 1:1 (0.2 ml), and BSA (6.9 mg) in H2O (0.4 ml). MALDI-
TOF-MS: 71341 (10-BSA).
We thank Prof. P. Potier, Gif-sur-Yvette, France, for a gift of DAB (1). Dr. J. Schmidt, Halle, for ESI-MS
analyses, and Dr. N. Oldham for recording MALDI-TOF mass spectra. Emily Wheeler, Jena, is gratefully
acknowledged for editorial and linguistic support in the preparation of the manuscript. This work was financially
supported by the European Community (AIR3-CT94-1979) and the Fonds der Chemischen Industrie (Frankfurt
a.M.).
REFERENCES
[1] M. E. Wall, M. C. Wani, in ꢁThe Alkaloids: Chemistry and Biologyꢂ, Ed. G. A. Cordell, Academic Press, San
Diego, 1998, Vol. 50, p. 509.
[2] Y. Guo, M. Jaziri, B. Diallo, R. Vanhaelen-Fastre, A. Zhiri, M. Vanhaelen, J. Homes, E. Bombardelli, Biol.
Chem. Hoppe-Seyler 1994, 37, 5281.
[3] P. G. Grothaus, T. J. G. Raybold, G. S. Bignami, C. B. Lazo, J. B. Byrnes, J. Immunol. Methods 1993, 158, 5.
[4] S. R. Svojanovski, K. L. Egodage, J. Wu, M. Slavik, G. S. Wilson, J. Pharm. Biomed. Anal. 1999, 20, 549.
[5] C. Bicamumpaka, M. Page, J. Immunol. Methods 1998, 212, 1.

Helvetica Chimica Acta ± Vol. 84 (2001)
1995
[6] A. E. Mathew, M. R. Mejillano, J. P. Nath, R. H. Himes, V. J. Stella, J. Med. Chem. 1992, 35, 145.
Ä
[7] A. A. Souto, A. U. Acuna, J. M. Andreu, I. Barasoain, M. Abal, F. Amat-Guerri, F. Angew. Chem. 1995,
107, 2910.
[8] V. Guillemard, C. Bicamumpaka, N. Boucher, M. Page, Anticancer Res. 1999, 19, 512.
Â
Â
[9] J.-D. Bourzat, A. Commercon, D. Guenard, F. Gueritte-Voegelein, P. Potier, European Patent, 1993, 524,
093.
[10] R. B. Greenwald, A. Pendri, D. Bolikal, J. Org. Chem. 1995, 60, 331.
[11] J. Kant, W. S. OꢂKeefe, S.-H. Chena, V. Faina, C. Fairchild, K. Johnston, J. F. Kadrow, B. H. Long, D. Vyas,
Tetrahedron Lett. 1994, 35, 5543.
[12] W. Mormann, S. Hoffmann, W. Hoffmann, Chem. Ber. 1987, 120, 285.
[13] L. Bhat, Y. B. Liu, S. F. Victory, R. H. Himes, G. I. Georg, Bioorg. Med. Chem. Lett. 1998, 8, 3181.
[14] J. P. Pulicani, D. Bezard, J. D. Bouchard, M. Zucco, D. Deprez, A. Commercon, Tetrahedron Lett. 1994, 35,
9717.
[15] B. F. Erlanger, Methods Enzymol. 1980, 70, 85.
[16] C. B. Reese, J. C. M. Stewart, Tetrahedron Lett. 1978, 4273.
Received February 21, 2001